Laboratory Study Of Different Ways Of Bone Marrow Mesenchymal Stem Cell Transplantation For Adriamycin - Induced Nephropathy In Rats

ObjectiveThe present study is aimed for isolation, culture and identification of the bone marrow mesenchymal stem cells (MSCs) in SD rats, to observe the safety of MSCs venous and arterial transplantation, and its distribution in the kidney of SD rat, to explore the feasibility of MSCs transplantation for treatment of chronic adriamycin nephropathy and analyze the possible mechanisms, providing theoretical and experimental basis of MSCs transplantation for clinical treatment of chronic adriamycin nephropathy.Methods1. Isolation, culture and identification of the bone marrow mesenchymal stem cells (MSCs) in rats. The femur, tibia and fibula were taken from 12-week male SD rats and placed in phosphate buffered saline (PBS). The marrow cavity was rinsed with 10% fetal bovine serum (L-DMEM) culture solution. The bone marrow cell suspension was collected and added with Ficoll separating medium. The precipitated bone marrow cells were collected after centrifugation for 30 min at 2000 r/min. The precipitates were pipetted with PBS buffer solution and then centrifuged for 10 min at 1600 r/min. The supernatant was discarded. The cells suspended in PBS solution were inoculated in a plastic culture flask and cultured in an incubator at 37 ℃ and 5% CO2. The cell phenotype antigens were detected by flow cytometry and differentiation potentials were observed by inducing to osteoblasts and fat cells. The bone marrow MSCs at P4-P6 were stained for labeling. They were digested with 0.1% trypsin, and then centrifuged after termination of digestion with the L-DMEM medium containing 10% FBS. The cell suspension was prepared with serum-free L-DMEM medium, and the cell concentration was adjusted to 2×106/ml. The in vitro labeling method of rat bone marrow mesenchymal stem cells:After in vitro BMSCs at P4-P6 reached up to 80% confluence, they were placed in 2% O2,93% N2 and 5% CO2 for 48 h to induce MSCs hypoxia. Ad5/F35 adenovirus carried with enhanced green fluorescent protein (EGFP) transfection solution was used for transfection. After 6 h of culture, the same amount of L-DMEN was added; and 24 h later, the expression of green fluorescence was observed under an inverted microscope. Flow cytometry was used for analysis of the expression of CXCR4 on the surface of MSCs.2. Exploration of the effects of different ways of MSCs transplantation on chronic adriamycin nephropathy in rats. Seventy-two 14-week male SD rats were randomly divided into six groups, with twelve in each group. ① Adriamycin model control group (ADR Group):SD rats underwent surgical removal of left kidney and intravenous injection of 4 mg/kg adriamycin. After the surgery, rats were allowed to be natural awake. A week later, rats were given intravenous injection of 4 mg/kg adriamycin via the caudal vein. ② Sham group:The surgical approach was similar to ADR group. When entered into the abdominal cavity, the left kidney was separated other than being removed. The incisions were sutured layer by layer. After the surgery, rats were naturally awake. Sham group underwent injection of the same amount of normal saline while ADR group underwent injection of adriamycin. ③ Adriamycin model + cellular transplantation via the caudal vein group (ADR + MSCs-V group): After the last injection of adriamycin, rats underwent intravenous injection of 0.5ml MSCs (2×106/ml) via the caudal vein at the fourth week, which was repeated at the fifth week. ④ Adriamycin model + cellular transplantation via the renal artery group (ADR + MSCs-A group):After the last injection of adriamycin, rats underwent intravenous injection of 0.5ml MSCs (2×106/ml) via the renal artery at the fourth week, which was repeated at the fifth week. ⑤ Sham + intravenous injection of MSCs via the caudal vein group (Sham + MSCs-V group):After the last injection of normal saline, rats underwent intravenous injection of 0.5ml MSCs (2×106/ml) via the caudal vein at the fourth week, which was repeated at the fifth week. ⑥ Sham + arterial injection of MSCs via the renal artery group (Sham + MSCs-A group):After the last injection of normal saline, rats underwent arterial injection of 0.5ml MSCs (2 ×106/ml) via the renal artery at the fourth weeks, which was repeated at the fifth week. Four rats were selected from six groups at 0 w,1 w and 2 w after the last cellular transplantation respectively. Their 24h urines were collected prior to execution. The blood specimens, kidney, heart, liver and bone marrow were collected for the following checks after death. ① The blood specimens were used for detection of hemoglobin, renal function (blood urea nitrogen and creatinine), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase-MB (CK-MB), serum albumin and serum sodium. ② The urine specimens were used for quantitative detection of 24h urinary protein. ③ At the second week after the cellular transplantation, the rat kidney tissues in ADR group, Sham group, ADR + MSCs-V group and ADR + MSCs-A group underwent hematoxylin-eosin (HE) staining, Masson staining and periodic acid-silver metheramine (PASM). Their morphological changes of renal tissues were observed under an optical microscopy. ④ The rat heart, liver and bone marrow in ADR group, Sham group, ADR + MSCs-V group and ADR + MSCs-A group were made pathological sections. Their morphological changes were observed under an optical microscopy. ⑤ The expressions of AQP1 and AQP2 in rat kidney in ADR group, Sham group, ADR + MSCs-V group and ADR + MSCs-A group were detected by immunohistochemistry. The ultrastructure of rat kidney in each group was observed under an electron microscopy.3. Eighty-four 14-week male SD rats were randomly divided into four groups, including ADR group, ADR + MSCs-V group, ADR + MSCs-A group, with twenty-four rats in each group, and Sham group with twelve rats. The preparation of model and the dose of MSCs were the same with the former. The MSCs cultured under hypoxic conditions and transfected with the EGFP transfection solution were used. Six rats were selected from ADR group, ADR + MSCs-V group and ADR + MSCs-A group at 1 d,7 d,14 d and 30 d after the transplantation of MSCs, respect-tively. Three rats were selected from Sham group. They were executed, and their kidneys were collected for the line following tests. ① The observation under a laser confocal fluorescence microscopy showed the distribution of MSCs labeled with green fluorescence in the kidney. ② Real-time PCR was used to detect the expression of SDF-lmRNA in the kidney. ③ At the 14th day after transplantation, Western- blotting was used to detect the level of SDF-1 protein in the kidney.4. Thirty-two 14-week male SD rats were randomly divided into four groups, including ADR group, ADR + MSCs-A group, ADR + NS-A group and Sham group, with eight rats in each group. The reparation of model and the dose of MSCs were as the same with the former. However, both the ADR + MSCs-A group and ADR + NS-A group underwent renal arteriography by using the renal artery approach through the carotid artery. The ADR + MSCs-A group was then given intubation and injection of MSCs, and the ADR + NS-A group was given intubation and injection of the same amount of normal saline (NS). Four rats were selected from four groups and executed at 1 d and 7 d after the last cellular transplantation. The blood samples and kidney were collected for the following tests. ① The blood specimens were used for the blood routine, serum urea nitrogen and serum creatinine examinations. ② The morphological changes of kidney tissues were observed under an optical microscope.Results1. The primary cells were adherent within 24-48 hours after enzymatic digestion. They were in faster growth after one week, showing a parallelly arranged growth. After two or three weeks, they formed an adherent cells layer at 70%　confluence. The uniform fusiform cells were obtained after repeated passaging. The cell phenotype was observed by using flow cytometry. It was found that CD29, CD44 and CD90 MSCs markers were expressed in these cells, but CD45 and CD11b were not expressed. The sample contained cells with an adipogenic phenotype when identified by oil red O staining after 14 days of adipogenic differentiation. The sample formed mineralized matrix when detected by the calcification of the matrix (von Kossa) after 14 days of osteogenic differentiation. Cells at P4 in fast growth were placed in 2%　O2, 93% N2 and 5% CO2 environment for 48 h to induce the hypoxia. They were then transfected with Ad5/F35 adenovirus carried with enhanced green fluorescent protein (EGFP) transfection solution. After being cultured for 6 h, they were added with the same amount of L-DMEN.24 h later, the expression rate of green fluorescence was observed as 75% under an inverted microscope, with no significant difference in the form from the cells cultured under normal conditions. The expression of CXCR4 was weaker on the surface of BMSCs cultured under normal conditions, but it was significantly enhanced on the surface of BMSCs cultured under hypoxic conditions.2. ① There were no significant differences in the weight gain, hemoglobin, blood urea nitrogen and serum creatinine levels at each time point between Sham + MSCs-V group and Sham + MSCs-A group (P>0.05). ② The kidney function (blood urea nitrogen and creatinine) and 24h urine protein quantitation in ADR group, ADR + MSCs-V group and ADR + MSCs-A group at each time point were significantly higher than the Sham group (P<0.05). At 0 w after the last injection of stem cells,24h urine microalbuminuria in ADR + MSCs-A group was reduced compared with ADR group (P＜0.01). At 1 w after the last injection of stem cells, the serum creatinine in ADR + MSCs-A group was reduced compared with ADR group and ADR + MSCs-V group (P＜0.01); 24 h urinary protein and 24 h urine microalbuminuria were significantly lower than ADR + MSCs-V group (P＜0.01). At 2 w, although the serum creatinine in ADR + MSCs-A group was lower than ADR group (P<0.01), it was not significantly different from MSCs-V group. ③ The serum albumin levels and 24h urine levels in ADR + MSCs-A group and ADR + MSCs-V group were significantly increased compared with ADR group (P<0.05); however, the serum sodium levels and 24h urine protein quantitation were significantly reduced compared with ADR group (serum sodium:P＜0.05; 24h urine protein quantitation: P＜0.01). There were no significant differences in above detection indexes between ADR + MSCs-A group and ADR + MSCs-V group. ④ There were no significant lesions in the glomerulus, renal tubule and renal interstitium. The glomerulus, renal tubules and renal interstitium in ADR group had varying degrees of damages. The pathological changes in renal interstitium inflammation in ADR + MSCs-A group and ADR + MSCs-V group were lighter than ADR group, with a smaller range. ⑤ Compared with ADR group, the serum albumin levels and 24h urine levels in ADR + MSCs-A group and ADR + MSCs-V group were increased (P<0.05); the serum sodium levels and 24h urine 24h urine protein quantitation were reduced (P＜0.05); the expressions of AQP1 and AQP2 in the kidney were significantly reduced (P＜0.05). The ultrastructure of rat kidney in ADR group showed degenerative changes, and MSCs transplantation improved the ultrastructure.3. ① AT the first day after transplantation of GFP-labeled MSCs, they were found to be distributed in the renal tubules in ADR + MSCs-V group. While in ADR + MSCs-A group, they were distributed in the renal tubules and also in the glomerulus. The distribution was gradually increased, and reached the maximum at the 14th day. ② The expression of SDF-1 in rat renal tissue in ADR group was significantly increased compared with Sham group (P＜0.05), and increased gradually over time. ③ After the transplantation of allogeneic bone marrow MSCs cultured under hypoxic conditions, the expression of SDF-1 was lower than ADR group, with statistically significant difference at 14th day (P＜0.05); but there was no significant difference between ADR + MSCs-A group and ADR + MSCs-V group.4. ① At the first day, the renal function indexes and hemoglobin levels in ADR + MSCs-A group and ADR + NS-A group were not significantly different from ADR group (P>0.05). At the 7th day, the renal function indexes and hemoglobin levels in ADR + MSCs-A group and ADR + NS-A group were also not significantly different from ADR group (P>0.05); but the levels of blood urea nitrogen and serum creatinine in ADR + MSCs-A group were significantly reduced (P＜0.05), and the hemoglobin level was significantly increased (P＜0.05). ② The renal pathology in rats under an light microscope showed that the degrees of injury of glomerulus, renal tubules and renal interstitium in ADR + NS-A group were equivalent to ADR group, but the pathological changes of renal interstitium inflammation were lighter in ADR + MSCs-A group, with a smaller range.Conclusions1. The cultured bone marrow mesenchymal stem cells were in line with the characteristics of stem cells. Under the hypoxic conditions, the expression of CXCR4 was increased on the surface of cells.2. It is safe to transplant mesenchymal stem cells among the same kind of bone marrow. Over a period of time after transplantation, the effect of MSCs transpla-ntation via renal artery was better than via peripheral vein. MSCs transplantation could reduce urine albumin excretion in rats, increase serum albumin levels, reduce blood sodium levels, and down-regulate the overexpressions of AQP1 and AQP2, thereby protecting the kidneys.3. In the renal tissues of rats with chronic adriamycin nephropathy, the increase in the expression of SDF-1 might be associated with the increase in the migration of bone marrow mesenchymal stem cells. The mechanism of bone marrow mesenchymal stem cells in the therapy of adriamycin nephropathy remained to be further studied.4. Renal arteriography combined with transplantation of mesenchymal stem cells could help to repair the renal injuries in rats with chronic renal failure. This method could simulate the way of clinical intervention of stem cell transplantation via the renal artery, which could be applied and promoted in experimental studies.