Research For The Mechanism Of Embryonic Stem Cells Differentiating Into Kidney Tubular Epithelial Cells In Vitro And The Role Of ES In The Repairment Of Impaired Kidney In Vivo

Background and Objective: The general application of antibiotics and antitumor drugs make kidney injury induced by drugs become one main disease that harm people's health. As the renal tubular epithelial cell has the ability of concentrating and reabsorbing the glomerular filtrate , they are vulnerable to toxic injury especially when the drug concentration in the cells were higher. When the degree of drug-induced kidney impairment is gently, the patho-apparence is acute nephric tubule injury;when the degree is heavy, the patho-apparence is acute tubular necrosis. As the kidney has certain self-recovery ability, the former could be cured by the symptomatic treatment, but the latter often develop into renal failure. In the end , the patient would die. At present, the most effective therapeutic tool of renal failure is renal transplantation. A lack of donors limits the number of patients who receive kidney transplantations, and most patients must undergo dialysis throughout the rest of their life. Despite outstanding progress in regenerative medicine and tissue engineering, no successful methods to regenerate renal tissue have been established. The development of an effective regenerative medicine for the kidney would therefore provide invaluable benefits.Embryonic stem (ES) cells are continuously growing stem cell lines with totipotency , originally isolated from the animal earlier embryonic inner cell mass of or primordial germ cells , could differentiated into the cells of three blastodermic layers. Embryonic stem cells could defferentiate into various kinds of cells in vitro or in vivo, they could differentiated into difinited kinds of cells in definited microenvironment. These distinguishing features make ES cells become the chief choice of donor sources for cell therapy. Up to now, it has made the significant advancement in the study of inducing ES cells to differentiate directionally. But there are no reports about inducing embryonic stem cells to differentiate into kidney epithelial cells and the correlative differentiation mechanism. Meanwhile, there is no reports ahout the therapeutical effects of ES cells in the kidney injury induced by drugs in vivo. We co-cultured embryonic stem cells with kidney epithelial cells to study whether embryonic stem cells could differentiate into kidney epithelial cells;subsequently we also investigate the mechanism of embryonic stem cells differentiation by adding different chemical or biological reagents to the culture media. In order to study the renovation effects of embryonic stem cells on kidney injury, we first set up the mouse kidney injury modelsby cisplatinum and treat these mice with embryonic stem cells to observe whether ES cells could cure acute tubular necrosis, and the differentiation state of embryonic stem cells in vivo. We lay particular emphasis on the review of ES repairment effects in order to fertilize the theory of turnover in acute renal tubular necrosis.Section One: Model Establishment of Embryonic Stem Cells Differentiating Into Kidney Epithelial Cells In VitroMethods: (1) The collection and preparation of conditioned media: a nonolayer of kidney epithelial cells (TCMK-1) was cultured with fresh base medium without LIF until confluence. Two-day conditioned medium was then centrifuged, filtered (0.22-um pore size filter), diluted 1:3 with fresh base medium without LIF, and used.(2) Establishing model of embryonic stem cells differentiating into kidney epithelial cells (TCMK-1) in vitro: Use the porous membrane of the cell culture inserts as a substitute for the basal membrane and plate EBs derived from ES cells and TCMK-1 on each side of it respectively, with direct cellular contacts may formed between cells of each side through the pores. The factors of TCMK-1 cells secretion could take effect on embryonic stem cells through the pores. This group serves as contact co-cultured group;Plate EBs derived from ES cells directly in a gelatin-coated plate and induce them with the conditoned media. This group serves as conditoned-media induced group;Plate EBs derived from ES cells directly in a gelatin-coated plate and culture them with base media without LIF. This group serves as freedly differentiating group. In 0, 4, 7,10, 13, 16, 19d, we detected the cell morphologic change with fluorescent microscope and the expression of kidney development relative genes and the expression of the kidney epithelial cells specific marker protein (Ksp-cadherin) in 0, 4, 8, 12, 16, 20, 24d with RT-PCR technology and immunofluorescence technology. Results: (1) The results of fluorescence microscope show that EBs begin to diffferentiate gradually with the elongation of culture time. ES cells in the contact co-cultured group begin to differentiate earlier than the other groups.(2) The results of RT-PCR show that the kidney development relative genes, i.e. Pax-2, Wnt-4 and Ksp-cadherin, do not express in the EBs. EBs began to express the Pax-2 mRNA when induced for 4 days in the cell-induced group, and the level of Pax-2 mRNA increases with the ES differentiation. But Pax-2 mRNA become to decrease when induced for 13 days. There is also the expression of Pax-2 mRNA in the conditioned media induced group, but the level islower than that in the contact co-cultured group (p<0.05). Results of RT-PCR when EBs were induced for 16 days show that the ksp-cadherin mRNA begin to express in the contact co-cultured group and the conditioned media induced group, but the level of the former is higher than that in the latter (p<0.05). We did not detect the expression of ksp-cadherin mRNA in the freely differentiating group. The results of immunofluorescence chemistry technology are coincident to the results of RT-PCR. Conclusion: We had successfully established the model of ES-GFP cells differentiating into kidney epithelial cells in vitro. In the course of normal emryogenesis, ES cells differentiate along different lineages and form complex 3D tissue and organ structures. The model that we established has simulated the three-dimension structure in vivo to investigate whether ES cells could differentiate into specific kinds of cells in the specific microenviroment. Using the porous membrane of the cell culture inserts as a substitute for the basal membrane and plating ES cells and TCMK-1 on each side of it respectively, the secretary factors of kidney epithelial cells could induce ES cells to differentiate along the kidney cells and express protein markers representing all of the early, middle and late differentiation state of kidney development. According to the reports of documents, kidney specific protein (Ksp-cadherin) is the a valuable marker in ES cells differentiating into kidney epithelial cells. ES cells begin to express the Ksp-cadherin mRNA and protein after co-cultured for 16d, and the level of which increases with the elongation of co-cultured days. We do not detect the any expression of Ksp-cadherin in the freely differentiating group. Our expriment results indicate that ES cells could differentiate into specific kinds of cells in the specific microenviroment, cell-cell contact play roles in the course of differentiation. These data could provide materials for the treatment of nephritis in clinical.Section Two: Research For Role of Wnt-4 Signal Transduction In Es Cells Differentiating Into Kidney Epithelial CellsMethods: we study the role of Wnt-4 signal transdution in Es cells differentiating into kidney epithelial cells with the model of inducing ES differentiation established in the section one. Expression of the important moculars in the Wnt signal transduction are detected in different induced days. We set up the co-culture system of ES cells and kidney epithelial cells, and add chemical materials LiCl to the media to promote theWnt signal transduction. This group serves as LiCl group.The media of another co-culture system is added anti-wnt-4 antibody that is the inhibitor of Wnt signal transduction. The experiment group is the co-culture system whose media without any chemical meterials or biological meterials. The detect indexes are as follows: (1) we detect the expression of Pax-2, Wnt-4, Frizzled-4, Frizzled-6 mRNA in EBs by RT-PCR and Real-time PCR technology in different days, i.e.3d, 5d, 7d, 9d, lid, 13d. The expression of Ksp-cadherin mRNA is also detected in 3d, 7d, lid, 15d, 19d, 23d. (2) observating the expression of the important composition of wnt signal transduction: we detect the expression of non-phosphorylated p - catenin by western blotting according to the expression time of Wnt-4 mRNA in the LiCl group, i.e. 5d, 7d, 9d, lid and 13d. (3) observating the effect of Wnt signal transduction on ES differentiating into kidney epithelial cells in vitro: we detect the expression of Ksp-cadherin protein in ES cells by immunohistofluorescence chemeistry technology after being induced for 15d, 19d, 23d. The differentiation rate of ES cells is detected by flow cytometry. Results: the experiment results of RT-PCR show that the expression time of pax-2, wnt-4, frizzled and ksp-cadherin of ES cells in the LiCl group are earlier than those in the other two groups (p < 0.01) . Pax-2 begins to express when ES cells are induced for 3 days in the LiCl group and decreases when ES cells are induced for 7 days. It begins to express when ES cells are induced for 5 days in the other two groups. Wnt-4 mRNA begins to express when ES cells are induced for 5, 7 and 11 days in the LiCl group, the Experiment group and the Anti-wnt-4 group respectively. The level of wnt-4 mRNA increases with the ES differentiation. The express time and change tendency of Frizzled-6 mRNA are similar to those of Wnt-4 mRNA. During the whole experimental period, neither Frizzled-4 mRNA nor its protein was detected in any group. Ksp-cadherin mRNA begins to express when ES cells are induced for 11 days and 15 days in the LiCl group and the Experiment group, respectively. And the level of it in the LiCl group is higher than that in the Experiment group (p<0.01). There is no expression of Ksp-cadherin mRNA in the Anti-wnt-4 group. (2) Results of western blotting show that the expression of non-phosphorylated P - catenin increases after wnt-4 gene begins to express. The expression tendency of it in the Experiment group is similar to that in the LiCl group, but the level in former is lower than that in latter (p<0.01). The level of non-phosphorylated P - catenin is lower than that in the other two groups, and the level decreased with the differentiation. (3) Results of immunofluorescencetechnology and flow cytometry show that the rate of ksp-cadherin positive cells in the LiCl group is higher than that in the Experiment group (p<0.01). Conclusion: we observe the ES differentiation state in different groups by inhibiting the degradation of (3 - catenin to promote the wnt signal transdution with LiCl and by inhibiting the wnt signal transdution with anti-wnt-4 antibody in order to analyze the role of wnt signal transduction in the ES cells differentiating into kidney epithelial cells. The results show that the rate of ksp-cadherin positive cells in the LiCl group is higher than that in the Experiment group. No ksp-cadherin positive cells are detected in the Anti-wnt-4 group. Thus we think that wnt-4 binds the receptor Frizzled-6 not Frizzled-4 to promote the canonical signal transdution and change the expression of P - catenin to play roles in the ES cells differentiating into kidney epithelial cells in vitro.Section three: Role of embryonic stem cells in the recovery of acute tubular necrosis in miceMethods: BALB/C female or male mice, a mo of age at the start of the experiments, were used. The medicine acute kidney injury is induced in mice by caudal vein injection of cis-diaminedichloroplatinum (cisplatin;12.7mg/kg) dissolved in sterile 0.9% saline solution. After establishing successfully the model, we injected the ES cells ,TCMK-1 cells and 0.9% saline solution by caudal vein respectively, serving as the ES cell treatment group , the TCMK-1 cell treatment group and the saline solution injection group. Several days later, we detect the mice kidney pathological change by HE staining, the BUN and Scr change in blood by blood detection and the expression of GFP by RT-PCR and immunofluorescence technology. Results: the results of HE staining show that there is severe tubular injury characterized by luminal ectasia, marked cytoplasmic simplification, cytoplasmic eosinophilia, loss of brush border, dropout of tubular epithelia, and multiple apoptotic figures in the mice kidney when injected for 4 days. The hematology detection results show that the level of BUN in blood of the experiment group is significantly lower than that of the control group after injecting for 4 days. GFP mRNA is detected by RT-PCR after injecting ES cells into the mice for 5 days, and the GFP protein is detected by immunofluorescence on 6 days. Conclusion: In this section, we establish successfully the mice acute kidney injury model induced by drugs and discover that embryonic stem cells also could differentiate into kidney epithelial cells in vivo. The kidney local microenviroment play important role in the ES cellsdifferentiating into kidney epithelial cells. Thus embryonic stem cells might become the important source for cell therapy of the acute kidney failure.Significance: 1. Our study first mimic the kidney microenviroment in vivo to induce embryonic stem cells to differentiate into kidney epithelial cells, providing the basis for studying the mechanism of kidney development. The establishment of this model and the study for the mechanism of ES cells differentiating into kidney epithelial cells must promote the acute kidney failure related clinical research, and facilitate the clinical application. 2. Our results show that Wnt-4 signal transduction play important role in the ES cells differentiating into kidney epithelial cells. We first find that Wnt-4 binds to the receptor FrizzIed-6 not Frizzled-4 and increase the expression of non-phosphorylated P - catenin to produce a marked effect. 3. We first study thetherapeutical effect of embyonic stem cells in the acute kidney induced by drugs,which would facilitate the embryonic stem cells serving as seed cells for the cell substitution therapy of renal failure.