Analysis of Structural and Functional Glomerular Changes in Hyperglycemic Rats That Treated with Hum

www.bacpl.org/j/pcc Physical Chemistry Communications, Volume 1 Issue 1, April 2014

Analysis of Structural and Functional Glomerular Changes in Hyperglycemic Rats That Treated with Human Umbilical Cord Blood Derived CD133 Negative Cells Hyperglycemic and Derived CD133 Negative Cells Farideh Feizi*1,3, Durdi Qujeq*1,2, Fariba Salmani2 *1

Cellular and Molecular Biology Research Center (CMBRC), Babol University of Medical Sciences, Babol, Iran

Department of Biochemistry and Biophysics, Faculty of Medicine, Babol University of Medical Sciences, Babol, Iran 2

Department of Anatomical Sciences, Faculty of Medicine, Babol University of Medical Sciences, Babol, Iran

3

*1

faridehfeizi@yahoo.com; *2dqujeq@hotmail.com; 3d.qujeq@mubabol.ac.ir

Received 9 February 2014; Revised 27 March 2014; Accepted 6 April 2014; Published 17 July 2014 © 2014 Biological and Chemical Publishing Abstract A number of recent studies have suggested that increasing blood glucose causes the glomerular hemodynamic changes .The purpose of the present investigation was to examine the effect of cord blood derived CD 133 negative cells on the structure and function of renal glomerular in hyperglycemic rats.

Experimental hyperglycemic was induced by injecting 120 mg/kg alloxan through the intraperitoneal within 10 min of preparation. CD133 negative cells were infused through the tail vein 7 days after the alloxan injection when blood glucose was more than 250 mg/dl. At the end of experiments rats sacrificed, The kidneys were weighed and their tissues were processed for light microscopy. Glomerular features were evaluated quantitatively using dissection as well as the Cavalieri method and were then compared with control groups. Our results demonstrated that in hyperglycemic rats, mild glomerular change accompanied was observed at 16 weeks of age hyperglycemic rats.

Keywords Derived CD133 Negative Cells; Glomerular Changes; Hyperglycemic; Rat

I. Introduction There exists increasing evidence of glomerular hemodynamic changes which contribute to the pathogenesis of diabetic nephropathy [1]. Previous studies have been reported on that diabetic nephropathy characterized by low‐ level excretion of albumin [2]. Also, diabetic nephropathy is characterized by the accumulation of extracellular matrix proteins in the glomerulus [3]. As reported by many investigators, four main hypotheses about how hyperglycaemia causes diabetic complications are: increased polyol pathway flux; increased advanced glycation end‐product formation; activation of protein kinase C isoforms; and increased hexosamine pathway flux [4]. There is evidence that diabetic nephropathy is characterized by an expansion of the glomerular mesangium [5]. In addition, previous studies establish that mesangial cells occupy a central anatomical position in the glomerulus, playing crucial roles in maintaining the structure and function of glomerular capillary tufts [6]. Based on recent investigators reports glomerular mesangium subject is important, because its accumulation correlates closely with renal impairment in diabetes [7–9]. Interestingly, it has been noted recently that high glucose concentrations have been shown to act on mesangial cells [10, 11]. Little attention has been paid to this issue in literature. The goal of this study is to investigate further the mechanisms hyperglycemic nephropathy.

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II. Materials and Methods Male rats (at 8–12 weeks of age, with 170‐230g) were purchased from Babol University of Medical animal center, housed in a animal room under controlled temperature (20‐25 ºC), and a 12‐h light/dark cycle with free access to water and food. The rats were maintained under standard conditions, and all animal work was carried out under protocols approved by the Institutional Animal Care and Utilization Committees of Babol University (No: 91/9/19‐ PJ301671).Rats were randomized into three groups (six rats per group): control rats, hyperglycemic rats, and hyperglycemic rats treated with CD133 negative cells. Experimental hyperglycemic was induced by injecting 120 mg/kg alloxan solubilized in sodium citrate buffer (pH 5.4) through the intraperitoneal within 10 min of preparation. Control rats were injected with an equivalent volume of sodium citrate buffer alone. CD133 negative cells were infused through the tail vein 7 days after the alloxan injection when blood glucose was more than 250 mg/dl. The effective dosage of CD133 negative cells was determined through preliminary experiments. Four, eight, twelve and sixteen weeks after the alloxan injection, blood was collected from all rats. All rats were anesthetized with an intraperitoneal injection of ether, and blood was collected.

1. Isolation of CD133 Negative Cells Human UCB cells were obtained from full‐term normal deliveries. Blood samples (n=4) from the umbilical cord were obtained from the Ayatollah Rohhani Hospital of Babol University, Babol, Iran. Written informed consent was obtained from the all mothers whose umbilical cord blood after a full explanation of the purpose and nature of all procedures was provided. The approval of the Ethics Committee of Babol University was obtained (No: 91919‐ 1671). Sample codes were (180342, 180480, 179362 and 180095). Each cord blood sample was collected into a 50 ml sterile polypropylene test tube containing 5 ml of citrate phosphate dextrose (Sigma, USA) as an anticoagulant. The mean volume of cord blood varied from 29 to 37 mL. Immediately after collection of UCB samples, approximately 10 mL of collected cord blood was mixed with the same volume of phosphate buffered saline (PBS). Units were placed in a 10 ml disposable centrifuge tube. An aliquot of 10 ml of this cell suspension was carefully layered onto the top of 10 ml of Ficoll‐paque TM premium (Sweden 17‐5442‐02) and subjected to density–gradient centrifugation (Centrifuge, Behdad; Iran) at 1700 rpm for 35 min. After 35 mines of centrifugation in a swinging bucket rotor, the intermediate layer was collected. Upon isolation, the cells of the mononuclear fraction were separated and counted (Hematology cell counter, Lc‐10, Austria micros).

Cord blood MNCs were isolated by a conventional centrifuge method through a Ficoll‐density gradient as described previously [12], in brief a whole –blood sample was layered onto the top of Ficoll (F5415,Sigma‐ Alderich ), which was followed by a centrifugation at 1300 g for 30 min. MNCs were recovered and washed twice with phosphate‐buffered saline (PBS, PH 7.4), ( 10010,Gibco ,invitrogen).

CD133 negative isolation was performed, as described previously [13, 14] by means of magnetic cell separation(MACS)columns (OctoMACS Acryl tube Rack, 130‐090‐448, 5100719002, Miltenyi Biotech, GmbH, MS Column Adapter , 130‐090‐543, 5100728182; MACS separation columns 130‐042‐201; sterile packed, 5ml‐Bottom tube 5 ml tubes for MS columns, sterile packed, 130‐091‐598) and antibodies recognizing the CD133 epitope according to the manufacturers procedure CD133 MicroBead Kit, 130‐050‐ Cells were passed through a Mini MACS column retained in a magnetic field, and the column was washed with PBS to remove unbound cells. The eluent contained CD133 negative cells, which were collected and used in subsequent experiments. After separation, cell populations were counted on an automatic cell counter (Sysmex‐1000i). For characterization of CD133 negative stem cells in bloods was determined using flow cytometry (PAS,Germany). Seven days after alloxan injection, rats were randomly divided into a control group, CD133 negative cell –treated group. The CD133 negative cells were centrifuged, re‐suspended with PBS, aggregation of the cells and to ensure reproducible deliver, CD133 negative cells were suspended in a large volume of phosphate buffer(100 μl) at a concentration of and transplanted to the rats through the tail vein. Animals were weighted before venous blood biochemical parameters concentrations were measured with kit. 16 weeks after treatment, the rats were sacrificed.

2. Isolation of Glomeruli Rats were sacrificed, and kidneys were rapidly removed and weighed. Right kidneys were then excised, decapsulated and immersed in the same fixative for 48 hours at room temperature. The fixed kidneys were

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embedded in 7% agar and sliced into 1 mm slices using a macrotome perpendicular to the longitudinal axis .Approximately 8‐10 slices were obtained from each kidney. The slices were arranged in a number of sequences on meshed tissue processing baskets and were then processed and embedded in paraffin. The blocks were systematically sectioned at 5 micrometers and every seventh section in order, with the first chosen randomly in the interval. They were then mounted on glass slides and stained with periodic acid‐Schiff (PAS). Every selected (1st and 7th) slice underwent a stereological analysis which involved the use of Olympus Optical BH2 microscopes.

To estimate the volume of cortex, medulla and the whole kidney, volume fraction (Vv) of the glomeruli, meangium and capillary. The physical dissector was used to estimate the numerical density of glomeruli and mesangial cells [15].

3. Assay for Biochemical Parameters Biochemical parameters including serum albumin, glucose and total protein were measured by kit (Pars azmoon, Tehran, Iran). Determination of albumin was performed based on its binding with the anionic dyes bromcresol green .Total protein was measured based on the biuret method.

4. Statistical Analysis All values have been presented as mean ± standard error. All tests were carried out in triplicate .Statistical analysis were done using SPSS version 18.0.The significance of differences between the mean values was determined by analysis of variance (ANOVA) or was determined using an unpaired two‐tailed Student’s t‐test, and a p value of less than 0.05 was considered statistically significant.

III. Results In contrast to animals only injected with citrate buffer, rats that received alloxan developed hyperglycemia. As shown in figure 1 morphometric measurements revealed no significant differences in the mean cortical volume of renal between hyperglycemic (375.78 ± 56.96 mm3)and were treated with CD133 negative cells (321.10 ±73.34 mm3),P>0.05.

450

mean Cortical volume (mm3)

400 350 300 250 200 150 100 50 0 group I

group II

FIG. 1 MEAN CORTICAL VOLUME. GROUP I, HYPERGLYCEMIC RATS; GROUP II, TREATED WITH CD133 NEGATIVE CELLS.

Mean density volume of glomerular in group II was less than group I, but difference was not significant (P>0.05).Also, mean density number of glomerular in group I was less than group II, but difference was not significant (P>0.05). Mean density volume of mesangium in group I was less than group II, difference was not significant (P<0.05). Mean density of glomerular capillaries volume in group II was less than group II, difference was not significant (P=0.023). The injection of CD133 negative cells after the induction of hyperglycemia effectively

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prevented decreasing albumin (2.35 ±0.28 vs.1.79 ±0.32, mg/dl, P<0.05) and total protein levels (5.89 ±0.87vs.3.89 ±0.8, mg/dl, P<0.05), respectively as shown in figures 3, 4. Blood glucose levels (figure 5) in alloxan‐induced hyperglycemic rats were significantly greater than those of non‐hyperglycemic controls (292.43 ±21.12 mg/dl). The increase in blood glucose was observed as early as one week after alloxan injection and was maintained throughout the 16 weeks study period. In contrast, alloxan‐ hyperglycemic rats treated with CD133 negative cells showed a substantial decrease in blood glucose levels compared with alloxan‐diabetic rats (100.22 ± 18.83 mg/dl); however, blood glucose levels remained greater in alloxan‐CD 133 negative cells rats than in non ‐hyperglycemic controls. Kidneys from untreated hyperglycemic rats at weeks 4‐16 contained many abnormal glomeruli (Fig. 6). In kidneys from CD133 negative cells treated diabetic rats mesangial cells number and mesangium volume were increased. The glomerular histological findings from PAS‐stained kidney sections of alloxan‐hyperglycemic and CD133 negative cells rats are shown in Fig 7.

mean density of glomerular volume (*10‐3 mm2)

4 3.5 3 2.5 2 1.5 1 0.5 0 group I

group II

FIG. 2 MEAN DENSITY GLOMERULAR VOLUME. GROUP I, HYPERGLYCEMIC RATS; GROUP II, TREATED WITH CD133 NEGATIVE CELLS.

3

Serum albumin level ( mg/dl)

2.5 2 1.5 1 0.5 0 group I

group II

FIG. 3 MEAN SERUM ALBUMIN LEVEL. GROUP I, HYPERGLYCEMIC RATS; GROUP II, TREATED WITH CD133 NEGATIVE CELLS.

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Serum total protein level ( mg/dl)

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8 7 6 5 4 3 2 1 0 group I

group II

FIG. 4 MEAN SERUM TOTAL PROTEIN LEVEL. GROUP I, HYPERGLYCEMIC RATS; GROUP II, TREATED WITH CD133 NEGATIVE CELLS.

Serum glucose level ( mg/dl)

350 300 250 200 150 100 50 0 group I

group II

FIG. 5 SERUM GLUCOSE LEVEL. GROUP I, HYPERGLYCEMIC RATS; GROUP II, TREATED WITH CD133 NEGATIVE CELLS.

FIG. 6 LIGHT MICROGRAPHS STAINED WITH PAS REAGENTS FROM ALLOXAN‐HYPERGLYCEMIC RATS (PAS; MAGNIFICATION, 400).

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FIG. 7 LIGHT MICROGRAPHS ALLOXAN‐HYPERGLYCEMIC RATS TREATED WITH CD133 NEGATIVE CELLS ,STAINED WITH PAS REAGENTS (PAS; MAGNIFICATION, 400).

IV. Discussion A growing body of clinical evidence clearly indicates the important role of hyperglycemia in the development of hyperglycemic nephropathy status. Up to now, a wide variety of strategies have been tested, with varying degrees of success. However, hyperglycemic nephropathy has been steadily increasing, and most of patients continue to show progressive renal injury. Therefore, an understanding of hyperglycemic‐induced molecular mechanisms is needed.

In this respect, stem cell therapy has become an attractive therapeutic strategy.

It is interesting that cord blood derivatives cells have shown great potential for therapeutic applications for various diseases including hyperglycemia [12].

Our first major observation was that hyperglycemia has been implicated in glomerular structure and function. Also, present results demonstrated that glomerular morophology were stained with PAS reagents different from hyperglycemic rats and rats treated with CD133 negative cells. Also, in agreement with these histological findings, the experimental results presented in this study demonstrated that albumin and total protein level increased in hyperglycemic status and decreased in treated with CD133 negative group. It is well known that little albumin is filtered through the kidney glomeruli. Also, albumin level is important for management and following up renal disease. The decrease of serum albumin, total protein and histological changes in 16‐weeks‐old hyperglycemic rats in our study was expected. These results are in agreement with those published by other investigators [16‐19]., However, the exact mechanism of CD133 negative cells on the renal cells remains unclear, and there is still an enormous of research need.

It suggests that the key pathogenic mechanisms involved in the induction and progression of hyperglycemic glomerular structure and function changes still remain active and unmodified by the present study.

Based on the present results and taken together with previous studies, we hypothesized that glomerular structure and function alterations could be due to the pathogenesis of hyperglycemic nephropathy through the excess glucose .

V. Conclusion The present results provide experimental evidence in support of the application of CD133 negative cells to minimize hyperglycemic‐induced side‐effects on the renal and improve the abnormal glucose metabolism. Also, these results suggest that the claimed beneficial effects of CD133 negative cells in hyperglycemic rats could be due

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to repair property of CD133 negative cells.

ACKNOWLEDGMENT

We express our gratitude to the staff of Department of Biochemistry, Babol University School of Medicine for their assistance in collection of blood and liver tissue samples. We thank Mr Shikhzadeh for excellent technical assistance. This investigation was a collaborative work between the Cellular and Molecular Biology Research Center and the Faculty of Medicine. The financial aid has been provided by Research Council of University. (No: 9031937). Also, this study was supported by the Research Council of Medicine faculty (p301‐11582.).

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