Physical Chemistry Communications, Volume 1 Issue 1, April 2014

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1 Physical Chemistry Communications, Volume 1 Issue 1, April 14 Analysis of Structural and Functional Glomerular Changes in Hyperglycemic Rats That Treated with Human Umbilical Cord Blood Derived CD1 Negative Cells Hyperglycemic and Derived CD1 Negative Cells Farideh Feizi *1,, Durdi Qujeq *1,, Fariba Salmani *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 Department of Anatomical Sciences, Faculty of Medicine, Babol University of Medical Sciences, Babol, Iran *1 faridehfeizi@yahoo.com; * dqujeq@hotmail.com; d.qujeq@mubabol.ac.ir Received 9 February 14; Revised 7 March 14; Accepted 6 April 14; Published 17 July 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 1 negative cells on the structure and function of renal glomerular in hyperglycemic rats. Experimental hyperglycemic was induced by injecting 1 mg/kg alloxan through the intraperitoneal within 1 min of preparation. CD1 negative cells were infused through the tail vein 7 days after the alloxan injection when blood glucose was more than 5 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 CD1 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 lowlevel excretion of albumin []. Also, diabetic nephropathy is characterized by the accumulation of extracellular matrix proteins in the glomerulus []. 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 [1, 11]. Little attention has been paid to this issue in literature. The goal of this study is to investigate further the mechanisms hyperglycemic nephropathy. 6

2 Physical Chemistry Communications, Volume 1 Issue 1, April 14 II. Materials and Methods Male rats (at 8 1 weeks of age, with 17 g) were purchased from Babol University of Medical animal center, housed in a animal room under controlled temperature ( 5 ºC), and a 1 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 PJ1671).Rats were randomized into three groups (six rats per group): control rats, hyperglycemic rats, and hyperglycemic rats treated with CD1 negative cells. Experimental hyperglycemic was induced by injecting 1 mg/kg alloxan solubilized in sodium citrate buffer (ph 5.4) through the intraperitoneal within 1 min of preparation. Control rats were injected with an equivalent volume of sodium citrate buffer alone. CD1 negative cells were infused through the tail vein 7 days after the alloxan injection when blood glucose was more than 5 mg/dl. The effective dosage of CD1 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 CD1 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: ). Sample codes were (184, 1848, 1796 and 1895). Each cord blood sample was collected into a 5 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 9 to 7 ml. Immediately after collection of UCB samples, approximately 1 ml of collected cord blood was mixed with the same volume of phosphate buffered saline (PBS). Units were placed in a 1 ml disposable centrifuge tube. An aliquot of 1 ml of this cell suspension was carefully layered onto the top of 1 ml of Ficoll paque TM premium (Sweden ) and subjected to density gradient centrifugation (Centrifuge, Behdad; Iran) at 17 rpm for 5 min. After 5 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 1, Austria micros). Cord blood MNCs were isolated by a conventional centrifuge method through a Ficoll density gradient as described previously [1], in brief a whole blood sample was layered onto the top of Ficoll (F5415,Sigma Alderich ), which was followed by a centrifugation at 1 g for min. MNCs were recovered and washed twice with phosphate buffered saline (PBS, PH 7.4), ( 11,Gibco,invitrogen). CD1 negative isolation was performed, as described previously [1, 14] by means of magnetic cell separation(macs)columns (OctoMACS Acryl tube Rack, , 51719, Miltenyi Biotech, GmbH, MS Column Adapter, , ; MACS separation columns 1 4 1; sterile packed, 5ml Bottom tube 5 ml tubes for MS columns, sterile packed, ) and antibodies recognizing the CD1 epitope according to the manufacturers procedure CD1 MicroBead Kit, 1 5 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 CD1 negative cells, which were collected and used in subsequent experiments. After separation, cell populations were counted on an automatic cell counter (Sysmex 1i). For characterization of CD1 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, CD1 negative cell treated group. The CD1 negative cells were centrifuged, re suspended with PBS, aggregation of the cells and to ensure reproducible deliver, CD1 negative cells were suspended in a large volume of phosphate buffer(1 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.. 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 7

3 Physical Chemistry Communications, Volume 1 Issue 1, April 14 embedded in 7% agar and sliced into 1 mm slices using a macrotome perpendicular to the longitudinal axis.approximately 8 1 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 BH 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].. 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..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.5 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 (75.78 ± mm )and were treated with CD1 negative cells (1.1 ±7.4 mm ),P> mean Cortical volume (mm ) FIG. 1 MEAN CORTICAL VOLUME. GROUP I, HYPERGLYCEMIC RATS; GROUP II, TREATED WITH CD1 NEGATIVE CELLS. Mean density volume of glomerular in was less than, but difference was not significant (P>.5).Also, mean density number of glomerular in was less than, but difference was not significant (P>.5). Mean density volume of mesangium in was less than, difference was not significant (P<.5). Mean density of glomerular capillaries volume in was less than, difference was not significant (P=.). The injection of CD1 negative cells after the induction of hyperglycemia effectively 8

4 Physical Chemistry Communications, Volume 1 Issue 1, April 14 prevented decreasing albumin (.5 ±.8 vs.1.79 ±., mg/dl, P<.5) and total protein levels (5.89 ±.87vs..89 ±.8, mg/dl, P<.5), respectively as shown in figures, 4. Blood glucose levels (figure 5) in alloxan induced hyperglycemic rats were significantly greater than those of non hyperglycemic controls (9.4 ±1.1 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 CD1 negative cells showed a substantial decrease in blood glucose levels compared with alloxan diabetic rats (1. ± 18.8 mg/dl); however, blood glucose levels remained greater in alloxan CD 1 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 CD1 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 CD1 negative cells rats are shown in Fig 7. 4 mean density of glomerular volume (*1 mm ) FIG. MEAN DENSITY GLOMERULAR VOLUME. GROUP I, HYPERGLYCEMIC RATS; GROUP II, TREATED WITH CD1 NEGATIVE CELLS. Serum albumin level ( mg/dl) FIG. MEAN SERUM ALBUMIN LEVEL. GROUP I, HYPERGLYCEMIC RATS; GROUP II, TREATED WITH CD1 NEGATIVE CELLS. 9

5 Physical Chemistry Communications, Volume 1 Issue 1, April 14 8 Serum total protein level ( mg/dl) FIG. 4 MEAN SERUM TOTAL PROTEIN LEVEL. GROUP I, HYPERGLYCEMIC RATS; GROUP II, TREATED WITH CD1 NEGATIVE CELLS. 5 Serum glucose level ( mg/dl) FIG. 5 SERUM GLUCOSE LEVEL. GROUP I, HYPERGLYCEMIC RATS; GROUP II, TREATED WITH CD1 NEGATIVE CELLS. FIG. 6 LIGHT MICROGRAPHS STAINED WITH PAS REAGENTS FROM ALLOXAN HYPERGLYCEMIC RATS (PAS; MAGNIFICATION, 4).

6 Physical Chemistry Communications, Volume 1 Issue 1, April 14 FIG. 7 LIGHT MICROGRAPHS ALLOXAN HYPERGLYCEMIC RATS TREATED WITH CD1 NEGATIVE CELLS,STAINED WITH PAS REAGENTS (PAS; MAGNIFICATION, 4). 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 [1]. 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 CD1 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 CD1 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 CD1 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 CD1 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 CD1 negative cells in hyperglycemic rats could be due 1

7 Physical Chemistry Communications, Volume 1 Issue 1, April 14 to repair property of CD1 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: 9197). Also, this study was supported by the Research Council of Medicine faculty (p ). REFERENCES [1] Sheridan A. M. Molecular Mechanisms Underlying Diabetic Nephropathy. Nephrology 4(8):1 6, 6. [] Molitch ME, Defronzo RA, et al. Nephropathy in diabetes. Diabetes Care 7(Suppl 1): S79 8,4. [] Osterby R, Gundersen HG, Horlyck A, Nyberg G, Westberg G: Diabetic glomerulopathy. Structural characteristics of the early and advanced stages. Diabetes :79 8, 198. [4] Brownlee, M. Biochemistry and molecular cell biology of diabetic complications. Nature 414:81 8,1. [5] Hey Jeong Noh, Hyun Chul Kim,Sang Sook Lee, Yu Na Kang, Young Mi Chae, Kwan Kyu Park: The Inhibitory Effect of sirnas on The High Glucose Induced Over expression of TGF 1 in Mesangial Cells. J Korean Med Sci 1: 4 45,6. [6] Dworkin LD, Ichikawa I, Brenner BM. Hormonal modulation of glomerular function. Am J Physiol 44: F95 14,198. [7] Monnier VM, Vishwanath V, Frank KE, Elmets CA, Dauchot P, Kohn RR: Relation between complications of type I diabetes mellitus and collagenlinked fluorescence. N Engl J Med 14:4 48, [8] Friedman EA: Advanced glycation end products in diabetic nephropathy. Nephrol Dial Transplant 14:1 9, [9] Friedman EA: Advanced glycosylated end products and hyperglycemia in the pathogenesis of diabetic complications. Diabetes Care :B65 B71, [1] Murphy G: Matrix metalloproteinases and their inhibitors. Acta Orthop Scand Suppl 66:55 6, [11] Nagase H, Woessner JF Jr: Matrix metalloproteinases. J Biol Chem 74: , [1] Qujeq D, Pourghasm M, Joursaraei G, Feiyzi F, Farsi M and Faraji AS. Mononuclear Derived from Human Umbilical Cord Normalize Glycemia in Alloxan Induced Hyperglycemic Rat. Cell Physiol Biochem 8: 8, 11. [1] Ruzicka K, Grskovic B, Pavlovic V, Qujeq D, Karimi A, Muller MM: Differentiation of human umbilical cord blood CD1+ stem cells towards myelo monocytic lineage. Clin Chim Acta. 4:85 9,4. [14] Grskovic B, Ruzicka K, Karimi A, Qujeq D, Müller MM: Cell cycle analysis of the CD1+ and CD1 cells isolated from Umbilical cord blood. Clin Chim Acta 4:17 178,4. [15] Gilbert JS, Lang AL, Grant AR, Nijland MJ. Maternal nutrient restriction in sheep: hypertension and decreased nephron number in offspring at 9 months of age. J Physiol. 565: ,5. [16] Shah R, Jindal R.M.: Reversal of diabetes in the rat by injection of hematopoietic stem cells infected with recombinant adenoassociated virus containing the preproinsulin II gene, Pancreatology : 4 48,. [17] Park J H, Park J, Hwang S H, Han H, Ha H.: Delayed Treatment with Human Umbilical Cord Blood Derived Stem Cells Attenuates Diabetic Renal Injury, Transplantation Proceedings, 44:11 116,1. [18] Park J H, Hwang I, Hwang S H, Han H, Ha H.: Human umbilical cord blood derived mesenchymal stem cells prevent diabetic renal injury through paracrine action, diabetes research and clinical practice 98: ,1. [19] Staniey N, Nahman JR, Karen L,Leonhart T, Fernado G. Costo, and Caoline L. HEBERT Effects of high glucose on cellular proliferation and fibronectin Kidney International, 41: 96 4,199.