Experimental Study Of Endothelial Progenitor Cells Transplantation Ameliorated Progressive Focal Segmental Glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is a group of clinical pathologic syndrome caused by various pathogenesis and mechanisms, with a pathological feature of focal and segmental glomerular solidification and scarring, and the clinical feature of nephrotic or non-nephrotic proteinuria. FSGS can be of primary renal diseases, or correlate with some other secondum diseases such as systemic lupus erythematosus, anaphylactoid purpura, acquired immunodeficiency syndrome, veinous drug abuse, glomerular hypertrophy, nephron reduction, etc.FSGS has high morbility and an increasing tendency. It is mainly treated by immune suppression and renin angiotensin system (RAS) blockage but without reparative regeneration of damaged glomerular, which usually causes progressive aggravation of renal function and chronic renal failure (CRF). Therefore, the exploration of new methods of delayed or ameliorated FSGS becomes the urgent demand and the focus of attention of the nephropathic field in the world.Studies in recent years consider that FSGS is a kind of podocyte diseases caused by some etiological factors that podocyte damage is the most important factor in the development of FSGS. (1) When podocytes are damaged and denudated, glomerular basement membrane will be exposed and this is commonly the earlier period of FSGS, which will cause glomerular capsular adhesion and sclerosis. (2) Damaged and exfoliated podocyte will transdifferentiate to macrophage, which will produce some fibrotic and sclerotic cytokine. (3) Damaged podocytes lose their physiologic functions, such as synthesis and secretion of vascular endothelial growth factor (VEGF). VEGF is synthesized in podocytes and will paracrine to VEGF receptors on jacent ECs which is the essential condition of EC proliferation and the glomerular capillary loop maintenance. VEGF also plays an important role in modulating capillary vessels in interstitium and peri-tubules. VEGF insufficiency will fail to keep the glomerular capillary loops normal and cause renal ischemia and hypoxia, which results in glomerular sclerosis and tubuar-linterstitial fibrosis. Together with podocytes damage, some other glomerular inherent cell damages occur, such as ECs. ECs damage will cause down regulation of VEGF receptors which forms the vicious cycle. At last the damages lead to CRF. In the vicious cycle, the podocytes recruitment, the VEGF reablement and the ECs recruitment may be the key point of amelioration of FSGS.Some studies showed that chronic renal insufficiency had endothelial progenitor cells (EPCs) dysfunction and intrarenal injection of bone marrow-derived EPCs could reduce podocytes and endothelial cell (EC) injury, so as mesangial cell activation in experimental glomerulonephritis. Therefore, it is supposed that EPCs and VEGF may play an important role in the podocyte and EC recruitment, which might be an effective method in ameliorating FSGS.Endothelial progenitor cells (EPCs), firstly reported by Asahara in 1997, are a group of precursor cells of ECs, which have not expressed mature EC phenotypes. EPCs could proliferate and differentiate into ECs. They take part in the vasculogenesis of human fetal vessels and exist in bone marrow, cord blood and peripheral blood with strong ability of promoting the angiogenesis after born. This conclusion updates the theory of traditional postnatal angiogenesis and vascular injury and repair, and offers some new thoughts for treatment of ischemic diseases. Recently, the studies on functions of EPCs on adult angiogenesis mainly focus on repairing of ischemic myocardium, ischemic limbs, injured corneal and skin, etc. With the development of isolation, cultivation, induction and amplification techniques, it is easy to obtain EPCs from peripheral blood or bone marrow. Thus in this experiment, EPCs derived from bone marrow were transplanted to progressive FSGS rats, or the recombinant adenovirus of VEGF165 were constructed in vitro and transfered to EPCs, then the gene tranfered EPCs were transplanted to progressive FSGS rats induced with Adriamycin injection. The progression of the FSGS were observed.Main Methods and Results1. Isolation, cultivation and identification of endothelial progenitor cells from rat bone marrowMononuclear cells were separated and purified from the male rats' bone marrow by density gradient centrifugation. After short-time cultivation and amplification under special inductive conditions, enough quantity of EPCs could be obtained in vitro, and finally, EPCs could differentiate into functional mature vascular endothelial cells with endothelial cell specific markers, which offered possibility for clinical applications.2. Construction of vascular endothelial growth factor 165 gene recombinant adenovirus and transfection of endothelial progenitor cellsVEGF165 gene was liberated from the vector of pcDNA3.1/VEGF165 via Xba I and Hind III digestion, and subcloned into shuttle vector of pAdTrack-CMV plasmid digested by Kpn I +HindIII, forming transfer vector of pAdTrack-CMV-VEGF165. Then pAdTrack-CMV-VEGF165 was linearized with Pme I and cotransformed into BJ5183 cells with adenovirus genomic plasmid of pAdEasy-1. The DNA of identified recombinant plasmid was digested with Pac I and transfected to 293 cells to package recombinant adenovirus particles. The polymerase chain reaction (PCR), sequencing and Western Blot analysis were used to detect target gene DNA and protein. The titre of Ad-VEGF165 was measured with the aid of green fluorescent protein (GFP) expression. EPCs derived from rat bone marrow were obtained by adherent culture for 6 days. After transfected by Ad-VEGF165 gene for 24h, the expression of VEGF protein was assessed using Western blot. Results: Positive recombinant bacterial clones were obtained after cotransformation of BJ5183 bacterial cells with pAdTrack-CMV-VEGF165 and pAdEasy-1 by method of CsCl₂. PCR test, Western Blot and sequencing analysis indicated that the recombinant adenovirus contained the inserted VEGF165. The titre of purified recombinant adenovirus was 1.4×10¹⁰ pfu/ml. After transfection, the expression of VEGF protein was significantly higher than that of untransfected EPCs group.3. Endothelial progenitor cells transplantation or VEGF 165 gene transfected endothelial progenitor cells transplantation ameliorated the progressive of focal segmental glomerular sclerosisFemale SD rats were divided randomly into normal control group (control group), adriamycin renal disease group (ADR group), EPC transplantation group (EPC group) and VEGF165 gene transfected EPCs transplantation group (VEGF group). ADR group, EPC group and VEGF group were carried out unilateral nephrectomy and injected with 5mg/kg and 3mg/kg of adriamycin via tail vein lweek(w) and 2w after operation, while the control group rats were carried out sham operation and then were injected with 0.9% Sodium Chloride solution of equal volume. After 5Gy X ray whole body irradiation were done 1w after the 2nd injection of Adriamycin, EPC group rats were transplanted with 1×10⁶ EPCs, and the VEGF group rats were transplanted with 1×10⁶ VEGF165 gene transfected EPCs via tail vein immediately, while the rats in control group and ADR group were only injected with 0.9% Sodium Chloride solution after whole body irradiation. The body weight and urine protein were measured before operation (0w) and 4w (1w after EPCs transplantation), 8w, 12w and 16w after nephrectomy. Y chromatosome incorporation was detected with in situ hybridization. The renal histological and ultrastructural changes were evaluated. VEGF, transforming growth factorβ1 (TGFβ1),α-smooth muscle actin (α-SMA) immunohistochemistry and Western Blot were detected. Results: (1) In situ hybridization showed that Y chromatosome positive cells incorporated in glomcrulus cells and tubular epithelial cells since 4^th week, and the incorporated positive cells could be found until the end of the experiment in EPC group and VEGF group. (2) Since the 4^th week, the body weight in ADR group, EPC group and VEGF group became lower than that in control group significantly; The rat weight in ADR group became lower than that in EPC group since 8w (p<0.05), but the VEGF group rats had the heveaiest body weight among the three groups. The proteinuria and SCr reached peak and were more severe in ADR group than those in EPC group, VEGF group and control group since the 4^th week, then decreased gradually, but from the 8^th week, the proteinuria and SCr were lower in VEGF group than that in EPC group. (3) Histological results showed that in ADR group, mesangial proliferation and the tubular-interstitial fibrosis were more remarkable than that in EPC group, VEGF group and control group. The image analysis showed that the index of glomerular sclerosis in ADR group was significantly higher than that in EPC group and VEGF group, the opening degree of glomerular capillary cavity in EPC group and VEGF group was significantly higher than that in ADR group. The situation was the same in capillary sectional area, but it was better in VEGF group than that in EPC group. The observation by electron microscope showed that the podocytes and tubular epithelial cells leisions in ADR group were more severe than that in EPC group and VEGF group since the 8^th week, and in the latter two groups, there was early reparation of podocytes and tubular epithelial cells. (4) Immunohistochemistry and Western Blot analysis showed that there were more VEGF but less TGF-β1 andα-SMA expression in VEGF group and EPC group than that in ADR group, meanwhile, there were the most expression of VEGF and lowest TGF-β1 andα-SMA expression in VEGF group.Conclusion1. After short-time cultivation and amplification under special inductive conditions, enough quantity of EPCs can be obtained in vitro, and will finally differentiate into functional mature vascular endothelial cells with endothelial cell specific markers. No immunologic rejection arises after autogenous implantation, which offers possibility for clinical applications.2. The method of homologous recombination in bacteria is a convenient and efficient method to conduct recombinant adenovirus and the conducted Ad-VEGF165 can effectively mediate target gene expression in cultured EPCs, which builds a sound foundation for further study.3. EPCs transplantation can ameliorate renal damages and may delay the progression of glomerular sclerosis and tubular-interstial fibrosis induced by adriamycin. VEGF165 gene transfected EPCs transplantation has a stronger effect of delaying the progression of glomerular sclerosis and tubular-interstial fibrosis induced by adriamycin.