| The incidence of end-stage renal disease in the world increased year by year,according to statistics,there are more than one million end-stage renal disease patients in china.Hemodialysis and kidney transplantation are two main treatment strategies for end-stage renal disease,however,hemodialysis can only partially replace renal function,and cause many com,lications,such as cardiovascular disease,bone disease,an so on.The long-term survival rate and the quality of life is low in dialysis patients.In addition,the high cost brings huge financial burdens on individuals,communities and health systems.Kidney transplantation is the best treatment of end-stage renal disease currently,but it is severely restricted by the shortage of kidney and immune rejection after transplantation.There are 1.2 million patients waiting for kidney transplantation in china,about 5,000 of them undergo kidney transplant each year,and a large number of patients are suffering or dying while waiting.Therefore,the research of kidney regeneration has drawn more and more social attention.In recent years,stem cell research has made great strides.Pluripotent stem cells have infinite proliferative capacity and multidirectional differentiation ability.They can differentiate into many cell types under certain conditions.Recent research shows that it is possible to induced embryonic stem cells differentiate into renal progenitor cells,including mesoderm(IM)cells and mesenchymal(MM)cells.Kidney progenitor cells can be induced to differentiate into 3D kidney structures characterized by tubules and glomeruli by co-culture with embryonic spinal cord in vitro.Therefore,pluripotent stem cells has a good prospect in kidney regeneration.To study renal regeneration,we must understand understand the process of kidney development and its molecular regulation mechanism.Mammalian kidney originates in the mesoderm,the cells from the outside of the body section to form a nephrotic cord,then the head of nephrotic cord developed into protonephros,central to mesonephros,the tail developed into metanephros.Protonephros and mesonephros gradually degenerate with the development of the embryo,metanephros eventually develops into adult kidneys.Kidney occurs from the pretubular aggregates.With the expression of Wnt9b,Pax8,Fgf8 and Wnt4,the pretubular aggregates rapidly form a cluster of approximately 30 cells.Subsequently,Wnt4 maintains the continuous expression of Pax8,Fgf8 and Lhx1,and converts the pretubular aggregates into an epithelial vesicle.Vesicles continue to develop into comma bodies and S bodies followed by the high expression of Brn1/Pou3f3 transcription factor,then mature into podocytes under the action of Wt1.The distal portion of the S-shaped body fuses with the collecting duct.At present,there are mainly six strategies for the study of kidney regeneration by stem cell:embryonic kidney transplantation,decellularized cadaveric scaffold,blastocyst complementation,direct differentiation,injection to xenoembryo,bioartificial kidney,etc.These six methods can regenerate renal cells,and some even can play partially renal function,but can not reproduce the complete kidney due to lack of vascularization,immune rejection,lack of cells and other reasons,and so on.In the blastocyst complementation method,the exogenous stem cells are injected into the blastocyst cavity in the blastocyst stage of the sall1-/-mouse so that the exogenous stem cells and the blastocyst develop together to form a chimera.This method can get the regenerating kidney derived from exogenous stem cells and the cell source can be controlled.The vasculature of the new kidney is derived from chimeras due to salll gene deletion does not affect vascular development,and the chimera animals pose a serious ethical issue.Directed differentiation of induced pluripotent stem cells/embryonic stem cells into kidney cells.Pluripotent stem cells can differentiate into any cell type in the body and self-assemble into tissues and organs.The stem cells are induced to differentiate into the renal cells by adding a variety of growth factors to the culture medium,and grow into a 3D structure of kidney tissue using the 3D culture method.The disadvantage is that the differentiation efficiency is low and vascularization can not be achieved.The latest research results show that organoids derived from PSCs in vitro are subcutaneously transplanted into the nude mice,and successfully develop vascularized functional "small kidney"which can be normal filtering blood and producing urine,which is an important milestone in the history of chronic kidney disease.Using the renal microenvironment to induce the differentiation of the exogenous stem cells into the renal cells,injecting exogenous stem cells into the developing position of the embryonic kidney so as to induce the exogenous cells differentiation into the chimeric kidney.However,the regeneration kidneys are fetal kidney,metanephros transplantation is still needed,and whole embryo culture in vitro is difficult,the chimeric kidneys still have greater immune rejection and ethical issues;The development of bioartifcial kidneys(BAKs)represents the intersection between regenerative medicine and renal replacement therapy,a renal tubule assist device(RAD)containing living renal proximal tubule cells has been successfully engineered,and it demonstrated differentiated absorptive,metabolic,and endocrine functions similar to normal kidneys in animal experiments in vitro and ex vivo.Compared to conventional blood filters and the extracorporeal blood circulatory system,RAD can maintain the viability of acute renal failure patients with uremia.In this study,we evaluate two kinds of methods for whole kidney regeneration.We summarize the pros and cons of embryonic kidney transplantation and decellularized cadaveric scaffold.The embryonic kidney has good differentiation potential.Fetal organs,such as the metanephros have been suggested as less immunogenic and more feasible for transplantation because(a)antigen-presenting cells that mediate direct host recognition of alloantigens and xenoantigens would be absent,(b)donor antigens such as majorhistocompatibility complex class ? and ? may not beexpressed by developing organs,(c)the immune response to transplanted fetal tissue differs from that to adult tissue in terms of the elicitation of a T helper 2-biased response when the target organ is of fetal origin,and(d)the vascular network of the transplanted embryo kidney will be derived from the receptor,which will reduce the immune response.Some studies have shown that metanephros transplantation can secrete erythropoietin(EPO)and renin;metanephros transplantation play a protective role on blood pressure in anephric rats with induced acute hypotension;metanephros transplantation inhibits the progression of vascular calcification in rats with adenine-induced renal failure;there are some studies comparing the site of transplantation in vivo,including the omentum,small intestine,abdominal aorta,renal artery,bladder,lymph nodes,but the renal capsule transplantation has not been reported,subretinal graft can make the graft and the host kidney together,(a)with the development of the kidney graft in vivo,it may fuse with the host kidneys;(b)the graft grow in the renal capsule,its collection system may be shared with the host kidney,if not,the host ureter anastomosis with the graft will be convenient;(c)compared to the omentum and abdominal aorta,renal capsule transplantation more easily,can significantly shorten the operation time,and do not need to open the abdomen.Therefore,the renal capsule as a new site of transplantation is worth further study.We dissected the metanephros from E15,and transplanted them into the omentum of unilateral nephrectomy rats(group A),and into subrenal capsule(group B),then compared their growth and development status and function.We found that(1)the contralateral kidney of unilateral nephrectomized rats significantly proliferated in both groups;(2)the two groups of grafts grew significantly after two weeks of transplantation;(3)the vascularization and the structures of glomerular,tubular were detected significantly by pathological examination,immunohistochemistry showed that PAX2,CD31 were positive,indicating that the grafts grew and vascularization well in vivo,OCT4,NANOG,SOX2 were negative,indicating that the kidneys have differentiated into mature renal cells,However,there was no significant difference between the two groups;4.urinary creatinine ELISA test showed that the graft has exerted renal function,there were not significant difference between the two groups.In summary,metanephroi transplantation can regenerate new kidney with all types of renal cells,after vascularization,the new kidney can exert partial renal function.There are no significant difference in the size of the graft or the degree of vascularization between omentum transplantation and subrenal transplantation,which indicate that the renal capsule can be used as a new site of metanephroi transplantation.Metanephroi transplantation can also play an important role in the maintenance of blood pressure and the inhibition of vascular calcification,so the metanephroi can be used as an important source of transplantable kidneys.In recent years,the tissue engineering technology based on cell biology and material science has developed rapidly.The three elements of tissue engineering are seed cells,biological materials,and the combination of cells and biological materials.At present,tissue engineering technology can be used to regenerate a variety of organizations with simple anatomical structure,such as bone,cartilage,blood vessels,bladder,etc.,which require uncomplicated vascular network when transplanted into the body.However,large organs with complex anatomical structure require a rigorous vascular supply system,such as heart,kidney,etc.The combination of simple synthetic materials with seed cells can not regenerate organs.Biological organ decellularized scaffolds provide more advanced biomaterials for tissue engineering and provide new hope for the regeneration of large organs.The decellularized scaffolds is an extracellular matrix 3D structure that removes all cellular components of an organ through physical,chemical,or a combination of both.The scaffold contains a large number of cytokines,which can provide microenvironment for seed cells to induce their directional differentiation.Acellular kidney scaffolds have been successfully produced in a variety of mammalian species such as rats,pigs,monkeys and the like.A research reported that acellular kidney scaffold can induce normal kidney to grow to inferior pole of kidney,when one third of inferior pole of normal kidney was cut off and make up with the acellular kidney scaffold after 4 weeks.In this study,we establish a platform of making rat renal acellular scaffolds with a variety of acellular cytotoxic agents,and then we chose human umbilical vein endothelial cells as seed cells,which is a pluripotent stem cells with the ability to differentiate into a variety of cell types.Human umbilical vein endothelial cells were perfused into rat renal acellular scaffolds through a continuous flow perfusion method,then,a new kidney with partial structure and function was regenerated.We examined the cell attachment and differentiation in scaffolds and assessed the function of regenerating kidneys,which provides a solid foundation for tissue engineering and kidney regeneration.Comparing and analyzing metanephroi transplantation and decellularized scaffolds,we found that metanephroi transplantation can regenerate all cell types of the adult kidney,and the cell number and the level of vascularization are superior to the method of the decellularized scaffold.However,there are still many unsolved problems.For example,there are ethical issues after being applied to human beings,and the long-term survival of the kidney graft.Decellularized scaffolds can regenerate normal-sized kidneys,which is more suitable for transplantation,the main limiting factors of this approach are inadequate cell attachment,uneven distribution,and thromboprophylaxis after transplantation.Both kidney regeneration methods have their own advantages,and one method which could offer all these advantages may be the optimal way for kidney transplantation. |
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