Transplant material and kidney transplant kit

Abstract

This transplant material is to be used in kidney xenotransplantation to a primate and contains a kidney anlage with bladder attached without including any blood vessels derived from a pig embryo.

Description

Transplant material and kidney transplantation kit The present invention relates to a transplant material and a kidney transplantation kit. This application claims priority based on Japanese Patent Application No. 2021-090066 filed in Japan on May 28, 2021, and the contents thereof are incorporated herein by reference. Heterologous donor organs have attracted attention as a means of avoiding the problem of donor shortage since organ transplantation was first performed. In particular, the development of porcine biotechnology has advanced, and genetically engineered pigs useful for xenotransplantation have been produced, and verification in preclinical models using non-human primates as recipients is underway (see Non-Patent Document 1). In recent years, genetically modified pigs have also been developed, and reports have been made on the achievement of long-term engraftment by control with very powerful immunosuppressive drugs (see Non-Patent Documents 2 to 4). Qi S, Peng J, Xu D, Vu MD, Liu D, Chen H. Improved techniques for kidney transplantation in the monkey. Microsurgery. 1999;19(7):335-7.Iwase H, Hara H, Ezzelarab M, et al. Immunologic and physiologic observations in baboons with life-supporting genetically engineered pig kidney grafts. Xenotransplantation. 2017;24(2):10.1111 / xen.12293.Higginbotham L, Mathews D, Breeden CA, et al. Pre-transplant antibody screening and anti-CD154 costimulation blockade promote long-term xenograft survival in a pig-to-primate kidney transplant model. Xenotransplantation. 2015;22(3):221-230. doi:10.1111 / xen.12166Kim SC, Mathews DV, Breeden CP, et al. Long-term survival of pig-to-rhesus macaque renal xenografts is dependent on CD4 T cell depletion. Am J Transplant. 2019;19(8):2174-2185. doi:10.1111 / ajt.15329 However, in kidney xenotransplantation from pigs to primates, it is very difficult to control the immune response with only clinically approved immunosuppressive drugs, and further improvement is required. Therefore, an object of the present invention is to provide a graft material and a kidney transplantation kit that can suppress the recipient's rejection reaction and enable long-term engraftment when transplanted into a primate. The present invention includes the following aspects. [1] A graft material for renal xenotransplantation into primates, comprising a kidney primordium with a bladder that does not contain blood vessels derived from porcine fetuses. [2] The graft material according to [1], wherein the kidney primordium with a bladder is derived from a porcine fetus at 4 to 6 weeks of gestation. [3] The graft material according to [1] or [2], wherein the kidney primordium with a bladder is derived from a wild-type porcine fetus. [4] The graft material according to any one of [1] to [3], wherein the kidney primordium with a bladder further contains nephron progenitor cells derived from humans. [5] The graft material according to any one of [1] to [4], which is used for a patient administered with an immunosuppressant based on an immunosuppression protocol. [6] The immunosuppression protocol is (1) Administering at least one immunosuppressant selected from thymoglobulin and anti-CD20 antibody, and (2) Administering at least one immunosuppressant selected from a co-stimulation signal inhibitor via CD28, a calcineurin inhibitor, an inosine monophosphate dehydrogenase inhibitor, a steroid, an anti-CD25 antibody, and an anti-CD40 antibody, The graft material according to [5], which includes. [7] The immunosuppression protocol further (3) Administering at least one anti-inflammatory agent selected from an anti-IL-6 receptor antibody and a TNF-α inhibitor, and / or (4) Administering at least one adjuvant selected from a platelet aggregation inhibitor, an anticoagulant, an antibacterial agent, a hematopoietic agent, a gastric acid secretion inhibitor, and an antiviral agent, The graft material according to [6], which includes. [8] The immunosuppression protocol is (1) Independently administering thymoglobulin and anti-CD20 antibody 2 to 3 days before transplantation, (2) Administering a co-stimulation signal inhibitor via CD28 every 7 days after transplantation, or immediately after transplantation and every 7 days, (3) Continuously administering a calcineurin inhibitor 9 days before transplantation, Continuously administer an inosine monophosphate dehydrogenase inhibitor starting 5 days before transplantation. Continuously administer a steroid starting on the day of transplantation. Administer an anti-IL-6 receptor antibody 1 to 3 times on the day before transplantation and approximately every 7 days after transplantation. Administer a TNF-α inhibitor every 3 to 4 days starting immediately after transplantation, and As an adjuvant, administer at least one agent selected from a platelet aggregation inhibitor, an anticoagulant, an antibacterial agent, a hematopoietic agent, a gastric acid secretion inhibitor, and an antiviral agent. The graft material according to any one of [5] to [7], comprising the above. [9] The immunosuppressive protocol is Administer an anti-CD20 antibody approximately 2 to 3 weeks before transplantation. Continuously administer an inosine monophosphate dehydrogenase inhibitor starting approximately 2 weeks before transplantation. Administer an anti-CD25 antibody on the day of transplantation and on the 4th day. Continuously administer a calcineurin inhibitor and a steroid starting approximately 1 week before transplantation, and As an adjuvant, administer at least one agent selected from a platelet aggregation inhibitor, an anticoagulant, an antibacterial agent, a hematopoietic agent, a gastric acid secretion inhibitor, and an antiviral agent. The graft material according to any one of [5] to [7], comprising the above.

[10] A kidney transplantation kit comprising the graft material according to any one of [1] to [4] and a combination of drugs used based on an immunosuppressive protocol.

[11] The immunosuppressive protocol is Administer at least one immunosuppressive agent selected from thymoglobulin and an anti-CD20 antibody, and Administer at least one immunosuppressive agent selected from a co-stimulation signal inhibitor via CD28, a calcineurin inhibitor, an inosine monophosphate dehydrogenase inhibitor, a steroid, an anti-CD25 antibody, and an anti-CD40 antibody. The kidney transplantation kit according to

[10] , comprising the above.

[12] The immunosuppressive protocol further (3) administering at least one anti-inflammatory agent selected from an anti-IL-6 receptor antibody and a TNF-α inhibitor, and / or, (4) administering at least one adjuvant selected from an antiplatelet agent, an anticoagulant, an antibacterial agent, a hematopoietic agent, a gastric acid secretion inhibitor, and an antiviral agent, The kidney transplantation kit according to

[11] , comprising the above.

[13] The immunosuppressive protocol (1) independently administering thymoglobulin and an anti-CD20 antibody 2 to 3 days before transplantation; (2) administering a costimulatory signal inhibitor via CD28 every 7 days after transplantation, or immediately after transplantation and every 7 days; (3) continuously administering a calcineurin inhibitor starting 9 days before transplantation; (4) continuously administering an inosine monophosphate dehydrogenase inhibitor starting 5 days before transplantation; (5) continuously administering a steroid starting on the day of transplantation; (6) administering an anti-IL-6 receptor antibody 1 to 3 times on the day before transplantation and approximately every 7 days after transplantation; (7) administering a TNF-α inhibitor every 3 to 4 days starting immediately after transplantation; and (8) administering, as an adjuvant, an antiplatelet agent, an anticoagulant, an antibacterial agent, a hematopoietic agent, a gastric acid secretion inhibitor, and an antiviral agent. The kidney transplantation kit according to any one of

[10] to

[12] , comprising the above.

[14] The immunosuppressive protocol (1) administering an anti-CD20 antibody approximately 2 to 3 weeks before transplantation; (2) continuously administering an inosine monophosphate dehydrogenase inhibitor starting approximately 2 weeks before transplantation; (3) administering an anti-CD25 antibody on the day of transplantation and on the 4th day; (4) continuously administering a calcineurin inhibitor and a steroid starting approximately 1 week before transplantation; and administering, as an adjuvant, at least one agent selected from a platelet aggregation inhibitor, an anticoagulant, an antibacterial agent, a hematopoietic agent, a gastric acid secretion inhibitor, and an antiviral agent; The kidney transplantation kit according to any one of

[10] to

[12] , comprising . According to the present invention, there can be provided a graft material and a kidney transplantation kit capable of suppressing a recipient's rejection reaction and achieving long-term engraftment when transplanted into a primate. These are the blood test data of each animal in the examples. (A) No. 200 (Experiment 1) (B) No. 335 (Experiment 1) (C) No. 323 (Experiment 2) (D) No. 219 (Experiment 2) These are photographs of the kidneys of neonates transplanted in Experiment 1. (A) At the time of transplantation (B) 13 days after transplantation These are photographs of fetal claw frogs transplanted in Experiment 1. (A) 13 days after transplantation (B) 27 days after transplantation These are representative photographs comparing the histopathological features of neonatal kidneys and fetal claw frogs in Experiment 1. (A)-(C): Neonatal kidneys, (D)-(F): Fetal claw frogs (13 days after transplantation), (G)-(I): Fetal claw frogs (27 days after transplantation), (A)(B)(D)(E)(G)(H): Masson's trichrome stained images, (C)(F)(I): CD3 These are photographs of fetal claw frogs transplanted in Experiment 2. (A) 33 days after transplantation, (B) 50 days after transplantation, (C) 79 days after transplantation These are representative photographs comparing the histopathological features of fetal claw frogs in Experiment 2. (A)-(C): 33 days after transplantation, (D)-(F): 50 days after transplantation, (G)-(I): 79 days after transplantation, (A)(B)(D)(E)(G)(H): Masson's trichrome stained images, (C)(F)(I): CD3 ≪Graft material≫ In one embodiment, the present invention provides a graft material for renal xenotransplantation into a primate, comprising a kidney primordium with bladder without blood vessels derived from a porcine fetus. The graft material of the present embodiment is preferably used to improve the renal function of a primate with reduced renal function. ≪Kidney primordium≫ The renal primordium refers to the kidney in the fetal stage, which corresponds to the metanephros in mammals. In fetal mammals, a tissue formed by integrating the bladder-ureter-metanephric tissue is formed. In this specification, this is referred to as the kidney primordium with bladder. The inventors named this fetal bladder-ureter-metanephric tissue the cloaca graft. That is, in this specification, the kidney primordium with bladder is also referred to as the cloaca graft. When the renal primordium is transplanted into the abdominal cavity of an animal, blood vessels invade the cloaca from the recipient, and development continues, producing urine. As will be described later in the examples, when a neonatal pig kidney with blood vessels and a kidney primordium with bladder derived from a fetal pig without blood vessels were respectively transplanted into cynomolgus monkeys as recipients, the immunogenicity of the fetal kidney primordium (kidney primordium with bladder derived from fetal pigs) in porcine-simian xenotransplantation was lower than that of the neonatal kidney. In neonatal kidney grafts with vascular anastomosis, acute rejection reactions were observed several weeks later. On the other hand, despite being administered the same immunosuppression protocol, the fetal kidney primordium of pigs (kidney primordium with bladder derived from fetal pigs) was spared from rejection and continued to develop for a long time, specifically for at least about 80 days. That is, it was found that the inflow blood vessels of the developing (also referred to as growing) kidney after transplantation are those of the recipient, and vascular endothelial dysfunction, which is the first target of xenograft rejection, i.e., one of the hyperacute rejection reactions, can be avoided by using a kidney primordium with bladder derived from fetal pigs without blood vessels. The kidney primordium with bladder is preferably derived from fetal pigs at 4 to 6 weeks (28 to 42 days) of gestation, more preferably from fetal pigs at 28 to 30 days of gestation. By using the kidney at such a gestational stage, it is easy to mature into renal tissue that produces urine without differentiating into other tissues. In addition, the blood vessels of the heterologous recipient flow into the fetal kidney during the transplantation process, and the expression of donor antigens tends to decrease at the developmental stage of the kidney. In one aspect, the kidney primordium with bladder is preferably derived from wild-type porcine fetuses. That is, in the transplantation of conventional porcine-derived kidney tissues into primates, porcine genetic modification had to be carried out to avoid the intense rejection reaction of the recipient. However, as will be described later in the examples, in the present invention, by using the kidney primordium with bladder derived from wild-type porcine fetuses without genetic modification as a transplantation material, suppression of acute rejection reaction was confirmed. As one aspect, it is preferable that the kidney primordium with bladder has not been genetically modified to avoid rejection in xenotransplantation. Examples of such genetic modification include methods known to those skilled in the art (for example, Xiao-Hua Yu et al., Clinica Chimica Acta 514 (2021) 15-23, or Lu et al., Frontiers in Immunology, January 2020, Volume 10, Article 3060). Specifically, they include α1,3-galactosyltransferase (GalT) knockout, CMP-N-acetylneuraminic acid hydroxylase (CMAH) knockout, β-1,4N-acetylgalactosaminyltransferase (β4GalNT2) knockout, human CD39 (hCD39) gene introduction, human CD46 (hCD46) gene introduction, human CD47 (hCD47) gene introduction, human CD55 (hCD55) gene introduction, human CD59 (hCD59) gene introduction, human CD200 (hCD200) gene introduction, human CD274 (hCD274) gene introduction, human thrombomodulin (hTBM) gene introduction, human tissue factor pathway inhibitor (hTFPI) gene introduction, human endothelial protein C receptor (hEPCR) gene introduction, human tumor necrosis factor alpha-induced protein-3 (hA20) gene introduction, human heme oxygenase 1 (hHO1) gene introduction, cytotoxic T-lymphocyte-associated protein-4-immunoglobulin (CTLA4-Ig) gene introduction, and MHC class II transactivator dominant negative (CIITA-DN) gene introduction. The recipient into which the transplantation material of the present embodiment is xenotransplanted is a primate, including humans, monkeys, gorillas, chimpanzees, etc. Examples of monkeys include rhesus monkeys, cynomolgus monkeys, etc. As an aspect of primates, Old World monkeys such as cynomolgus monkeys or humans can be mentioned. As an aspect of primates, primates that do not express galactose-α.1,3-galactose can be mentioned. From the perspective of establishing the urinary excretion system, it is preferable that the bladder of the kidney primordium with bladder of the transplantation material of the present embodiment be used for transplantation so as to be connected to the ureter of the recipient. The kidney primordium with bladder may further contain nephron progenitor cells derived from humans. Examples of nephron progenitor cells include cells differentiated from pluripotent stem cells such as iPS cells and ES cells. The nephron progenitor cells may be patient-derived cells or cells of another person with suppressed rejection reaction (see Fujimoto T, Yamanaka S, Tajiri S, et al. Generation of Human Renal Vesicles in Mouse Organ Niche Using Nephron Progenitor Cell Replacement System. Cell Rep. 2020;32(11):108130. doi:10.1016 / j.celrep.2020. Article No. 108130). Human-derived nephron progenitor cells are injected into the kidney primordium of fetal pigs. By using a kidney primordium with bladder that further contains human-derived nephron progenitor cells, a chimeric fetal kidney in the recipient grows. It is preferable that the size of the organ in the transplantation material be homologous to the organ originally possessed by the recipient. Therefore, for example, in order to form a kidney that can exhibit sufficient function in humans, it is preferable that the donor be a mammal having a size similar to that of a human organ. However, it is not necessary to have exactly the same size. If it is a kidney, as long as it has one-tenth of the overall function, sufficient dialysis can be performed and sufficient life can be maintained. For this reason, in the transplantation material of the present embodiment, the donor used is a pig, and it is judged that the size of the miniature pig's organ is sufficient. Next, the kidney primordium with bladder prepared as described above is transplanted into the abdominal cavity of the recipient. The transplantation site is not limited as long as it is within the abdominal cavity, and examples include the greater omentum, para-aortic region, etc. The greater omentum is the peritoneum that hangs down like an apron from the lower side of the stomach. Transplantation of kidney primordia into the greater omentum, para-aortic region, etc. of primates including humans can be performed by ordinary surgical procedures. For example, a method of picking up the kidney primordium to be transplanted with sharp forceps, making a slight incision on the surface of the adipose tissue of the greater omentum with the tip of the forceps, and embedding the tissue therein can be mentioned. In addition, transplantation can also be performed on the greater omentum, para-aortic region, etc. using an endoscope. ≪Kidney Transplantation Kit≫ In one embodiment, the present invention provides a transplantation method using the above transplantation material based on an immunosuppressive protocol, that is, the above transplantation material used based on an immunosuppressive protocol. In addition, in one embodiment, the present invention provides a kidney transplantation kit including the above transplantation material and a combination of drugs used based on an immunosuppressive protocol. Examples of the combination of drugs used based on an immunosuppressive protocol include a combination of an immunosuppressant, an anti-inflammatory agent and / or an adjuvant. In this specification, an immunosuppressant means a drug that suppresses the generation, activation or proliferation of immune cells. Specific examples of immune cells include lymphocytes such as B cells or T cells. Examples of immunosuppressants include drugs that damage T cells, drugs that inhibit the synthesis of T cells, drugs that suppress the activation of T cells, or drugs that suppress the differentiation (synthesis) / proliferation of T cells or the activation / proliferation of B cells. In addition, drugs that indirectly suppress the generation, activation or proliferation of immune cells, or drugs such as anti-inflammatory agents that suppress cytokine secretion and activity by the action of immune cells are also within the category of immunosuppressants. Specific examples of drugs that damage T cells or B cells include thymoglobulin that damages T cells, anti-CD20 antibodies that damage B cells (for example, rituximab, obinutuzumab). As agents that suppress the differentiation, activation or proliferation of T cells, specifically, substances that inhibit CD28 co-stimulation signals (for example, abatacept containing the extracellular domain of CTLA4 that binds to CD80 / CD86), calcineurin inhibitors (for example, tacrolimus, cyclosporine), inosine monophosphate dehydrogenase inhibitors (for example, mycophenolic acid, its ester mycophenolate mofetil (MMF), azathioprine, mizoribine), steroids (for example, methylprednisolone which is a glucocorticoid steroid), anti-CD25 antibodies (for example, basiliximab), or anti-CD40 antibodies (for example, iscalimab (CFZ533)) and the like can be mentioned. As anti-inflammatory agents, IL-6 inhibitors (for example, tocilizumab which is an anti-IL-6 receptor antibody, or sarilumab), TNFα inhibitors (for example, etanercept which is a soluble TNFα receptor, or adalimumab, golimumab or certolizumab pegol which are anti-TNFα monoclonal antibodies) and the like can be mentioned. As adjuvants, platelet aggregation inhibitors (acetylsalicylic acid (aspirin) etc.), gastric acid secretion inhibitors (famotidine etc.), anticoagulants (low molecular weight heparin, or nafamostat etc.), antibacterial agents (for example, penicillin antibiotics, cephem antibiotics, or sulfonamides etc.), hematopoietic agents (erythropoietin, or roxadustat etc.), antiviral agents (valganciclovir, or ganciclovir etc.) and the like can be mentioned. The administration methods of these agents are not particularly limited and may be appropriately determined based on an immunosuppressive protocol. For example, tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, or emulsions etc. are orally administered. Also, injections are intravenously administered alone or mixed with normal replenishing fluids such as glucose and amino acids, and furthermore, if necessary, are administered intraarterially, intramuscularly, intradermally, subcutaneously or intraperitoneally. Suppositories are rectally administered. Topical skin agents are applied, affixed or sprayed on the affected area. The dosages of these drugs vary depending on the patient's symptoms, weight, age, gender, etc. and cannot be determined unconditionally. In the case of oral administration, for example, an effective ingredient of 1 μg to 10 g per day, for example, 0.01 to 2000 mg per day may be administered. In the case of an injection, for example, an effective ingredient of 0.1 μg to 1 g per day, for example, 0.001 to 200 mg per day may be administered. In the case of a suppository, for example, an effective ingredient of 1 μg to 10 g per day, for example, 0.01 to 2000 mg per day may be administered. There are no particular limitations on the immunosuppressive protocol in this specification, that is, the combination of immunosuppressive agents and the timing of administration. As an example, (1) At the time of induction of immunosuppression, administer a relatively strong immunosuppressive agent, for example, a drug that damages immune cells, and (2) During the maintenance period of immunosuppression, administer an immunosuppressive agent with less concern about safety due to long-term administration for a certain period or longer, a drug that suppresses the differentiation (synthesis), activation or proliferation of immune cells, and / or an anti-inflammatory agent. It includes. During the induction period, it can be administered once or multiple times at an arbitrary time between about 2 weeks before transplantation and several days after transplantation (for example, on the day of transplantation to the 4th day). During the maintenance period, it can be continuously administered (for example, once a day, or once every two days, etc.) between about 2 weeks before transplantation and a certain period after transplantation. As the immunosuppressive protocol in this embodiment, (1) Administer at least one immunosuppressive agent selected from thymoglobulin and anti-CD20 antibody, and (2) Administer at least one immunosuppressive agent selected from a co-stimulation signal inhibitor via CD28 (for example, abatacept), a calcineurin inhibitor (for example, tacrolimus, rapamycin), an inosine monophosphate dehydrogenase inhibitor (for example, mycophenolate mofetil (MMF)), a steroid (for example, methylprednisolone), an anti-CD25 antibody, and an anti-CD40 antibody. It includes those containing. As one aspect, in the above (2), it is preferable to administer at least one immunosuppressant selected from a costimulatory signal inhibitor via CD28 (for example, abatacept), a calcineurin inhibitor (for example, tacrolimus or rapamycin), an inosine monophosphate dehydrogenase inhibitor (for example, mycophenolate mofetil (MMF)), a steroid (for example, methylprednisolone), and an anti-CD25 antibody. Such a protocol further (3) administering at least one anti-inflammatory agent selected from an anti-IL-6 receptor antibody and a TNF-α inhibitor, and / or (4) administering at least one adjuvant selected from a platelet aggregation inhibitor, an anticoagulant, an antibacterial agent, a hematopoietic agent, a gastric acid secretion inhibitor, and an antiviral agent. As one aspect of the immunosuppression protocol, (1) independently administering thymoglobulin and an anti-CD20 antibody 2 to 3 days before transplantation; (2) administering a costimulatory signal inhibitor via CD28 every 7 days after transplantation, or after transplantation and every 7 days; (3) continuously administering a calcineurin inhibitor starting 9 days before transplantation; (4) continuously administering an inosine monophosphate dehydrogenase inhibitor starting 5 days before transplantation; (5) continuously administering a steroid starting on the day of transplantation; (6) administering an anti-IL-6 receptor antibody 1 to 3 times on the day before transplantation and approximately every 7 days after transplantation, specifically, on the 7th day and the 14th day; (7) administering a TNFα inhibitor (for example, etanercept) every 3 to 4 days immediately after transplantation; and (8) administering a platelet aggregation inhibitor, an anticoagulant, an antibacterial agent, a hematopoietic agent, a gastric acid secretion inhibitor, and an antiviral agent as adjuvants. include those containing. As a specific protocol, (1) independently administering thymoglobulin and rituximab 2 to 3 days before transplantation; Administering abatacept every 7 days after transplantation, or on the days after transplantation and every 7 days; Continuously administering tacrolimus starting 9 days before transplantation; Continuously administering MMF starting 5 days before transplantation; Continuously administering methylprednisolone starting on the day of transplantation; Administering tocilizumab on the day before transplantation, and on the 7th and 14th days after transplantation; Administering etanercept every 3 to 4 days starting immediately after transplantation, and Administering at least one agent selected from aspirin, low molecular weight heparin, antibacterial agents, erythropoietin, and valganciclovir as an adjuvant; include those containing the above. Also, as one aspect of the immunosuppressive protocol, Administering an anti-CD20 antibody approximately 2 to 3 weeks before transplantation; Continuously administering an inosine monophosphate dehydrogenase inhibitor starting approximately 2 weeks before transplantation; Administering an anti-CD25 antibody on the day of transplantation and the 4th day; Continuously administering a calcineurin inhibitor and a steroid starting approximately 1 week before transplantation, and Administering a platelet aggregation inhibitor, an anticoagulant, an antibacterial agent, a hematopoietic agent, a gastric acid secretion inhibitor, and an antiviral agent as an adjuvant; include those containing the above. Also, as a specific protocol, Administering rituximab approximately 2 to 3 weeks before transplantation; Continuously administering MMF starting approximately 2 weeks before transplantation; Administering basiliximab on the day of transplantation and the 4th day; Continuously administering tacrolimus and methylprednisolone starting approximately 1 week before transplantation, and (5) Administering at least one agent selected from a platelet aggregation inhibitor, an anticoagulant, an antibacterial agent, a hematopoietic agent, a gastric acid secretion inhibitor, and an antiviral agent as an adjuvant. Those containing the same are exemplified. ≪Transplantation method≫ In one embodiment, the present invention provides a method for transplanting a kidney, comprising: step (a) of transplanting a transplantation material containing a bladder-equipped kidney primordium without blood vessels derived from a porcine fetus into a patient's body; and step (b) of connecting the bladder derived from the porcine fetus and the patient's ureter at a predetermined period after transplantation. The method of this embodiment can also be said to be a method for treating kidney diseases. As the transplantation material, those described above can be used. By leaving the transplantation material transplanted in step (a) for a predetermined period, the transplantation material grows and starts urine production. The predetermined period may be a period until the transplantation material grows sufficiently and before hydronephrosis occurs. The predetermined period may be appropriately adjusted according to the patient's symptoms, etc., but may be, for example, 1 to 10 weeks. Subsequently, step (b) is carried out at a predetermined period after transplantation. In step (b), the bladder of the transplantation material is incised, and this bladder is connected to the patient's ureter. Examples of the connection method include end-to-end anastomosis, end-to-side anastomosis, side-to-side anastomosis, and side-to-end anastomosis, and end-to-side anastomosis is preferred. By connecting the bladder of the transplantation material and the patient's bladder via the patient's ureter, the urine produced by the transplantation material can be excreted into the patient's bladder. More specifically, the urine produced by the kidney of the transplantation material is excreted to the bladder of the transplantation material, and then excreted from the bladder of the transplantation material to the patient's bladder through the patient's ureter. That is, as one embodiment of the present invention, there is provided a transplantation material containing a porcine fetal bladder-equipped kidney primordium used in the above-described kidney transplantation method and kidney disease treatment method. Also, as one embodiment of the present invention, there is provided the use of a porcine fetal bladder-equipped kidney primordium used in the above-described kidney transplantation method and kidney disease treatment method for the production of a transplantation material. The above transplantation method is preferably carried out together with the implementation of the above-described immunosuppression protocol. The immunosuppression protocol is as described in the above [6] to [9].

[0011] Any of the immunosuppressive protocols described in the present specification, such as ~

[0014] , may be used. In one embodiment, the present invention provides a transplantation material containing a porcine fetal kidney primordium with a bladder, and a combination of the transplantation material and a drug used based on an immunosuppressive protocol, which are used in the above-described method for transplanting a kidney or treating a kidney disease performed together with the implementation of the immunosuppressive protocol. Also, as one embodiment of the present invention, there is provided the use for manufacturing a kidney transplantation kit containing a transplantation material containing a porcine fetal kidney primordium with a bladder, and a combination of the transplantation material and a drug used based on an immunosuppressive protocol, which are used in the above-described method for transplanting a kidney or treating a kidney disease performed together with the implementation of the immunosuppressive protocol. The above transplantation method can be performed after implementing plasma exchange of the recipient in order to suppress the rejection reaction of the recipient (patient) against the transplantation material. In one embodiment, the present invention provides a method for transplanting a kidney or treating a kidney disease, comprising: a step (a1) of performing plasma exchange of a recipient (patient); a step (b1) of transplanting a transplantation material containing a kidney primordium with a bladder not accompanied by blood vessels derived from a porcine fetus into the body of the patient; and a step (b2) of connecting the bladder derived from the porcine fetus and the ureter of the patient after a predetermined period from the transplantation. That is, as one embodiment of the present invention, there is provided a transplantation material containing a porcine fetal kidney primordium with a bladder, which is used in the above-described method for transplanting a kidney or treating a kidney disease. Also, as one embodiment of the present invention, there is provided the use of a porcine fetal kidney primordium with a bladder for manufacturing a transplantation material, which is used in the above-described method for transplanting a kidney or treating a kidney disease. As methods of plasma exchange, double filtration plasmapheresis (DFPP), simple plasmapheresis (PE), plasma adsorption (PA), immunoadsorption (IAPP), etc. are known. It is preferable that plasma exchange before transplantation and the implementation of the above-described immunosuppressive protocol are both performed. Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to the following examples. [Abbreviations] TAC: Tacrolimus MMF: Mycophenolate mofetil [Experiment 1] Wild pigs were used as donors, and frozen 28 - 30-day-old fetal croakers were thawed and used as fetal kidneys. Also, 20 - 28-day-old neonatal pig kidneys weighing 5.0 - 6.12 kg were used as donor kidneys. The experiment was carried out in accordance with the appropriate guidelines for animal experiments. [Transplantation of neonatal pig kidneys and fetal croakers] Transplantation of neonatal pig kidneys with vascular anastomosis was carried out according to the method described in Non-Patent Document 2. The renal blood flow of the neonatal pig kidney donor was blocked after a midline abdominal incision. Subsequently, after reflow with a preservation solution containing 465 mL of Euro-Collins solution, 35 mL of 50% glucose solution, and 1,000 units of heparin, the kidney was removed together with the blood vessels and ureter and transported to the recipient facility for transplantation preparation (total storage time, 3 hours). All recipient monkeys were fasted from the evening before the surgery until the surgery. Maintenance anesthesia was administered using inhaled isoflurane (0.5% - 2%) after introducing atropine (0.1 mg / kg i.m.) and ketamine (10 mg / kg i.m.) on the day of the surgery. Also, butorphanol (0.1 mg / kg i.m.) was used as an analgesic, and potassium benzylpenicillin 47,500 units / individual was used as an antibacterial drug before and after the surgery. Laparotomy was performed on the above-pretreated recipient monkeys through a midline abdominal incision under general anesthesia. After ligating the left renal artery / vein and the left ureter, the left kidney was removed, and the neonatal pig kidney was placed in situ, and the blood vessels and ureter were anastomosed. Next, the retroperitoneum near the aorta and the left kidney was bluntly dissected to form a pocket, and two fetal cloacas (cloaca graft (kidney primordium with bladder)) were transplanted into the pocket of each animal using a spatula. After transplantation, the pocket was closed with LIGACLIP (registered trademark). Similarly, two fetal cloacas were transplanted into the greater omentum, and the pocket was closed with LIGACLIP (registered trademark). As a result, since it was covered with greater omentum tissue, the cloaca was prevented from falling off. After transplanting the cloaca around the aorta and into the greater omentum, the abdomen and the wound were closed, and the surgery was completed. [Recovery of transplanted tissue] Thirteen days after transplantation, the abdomen was reopened by a midline laparotomy. After laparotomy, the greater omentum was exposed, and the kidney derived from the transplanted fetal cloaca was identified using the clipped part with LIGACLIP (registered trademark) as a guide. A part of the kidney derived from the transplanted fetal cloaca was dissected together with the surrounding greater omentum tissue to avoid damage, and the tissue was collected. Subsequently, the intestine was manually moved to expose the transplanted neonatal pig-derived kidney. The area around the left renal artery / vein and the left ureter was dissected, and the abdominal aorta and the inferior vena cava were exposed. The left renal vein and the left ureter were ligated, the transplanted neonatal pig-derived kidney was incised on the porcine renal artery and vein side, and the kidney was removed en bloc. Finally, the abdomen and the wound were closed, and the surgery was completed. Administration of the immunosuppressant was continued even after the surgery while observing the dietary intake status. Furthermore, the remaining kidneys derived from the fetal cloacas transplanted into the greater omentum were identified again using the same method 27 days after transplantation, and the transplanted tissue was collected. [Experiment 2] [Fetal kidney transplantation] The preoperative and postoperative preparations of the recipient's fetal bladder were performed in substantially the same manner as in Experimental Example 1. The entire abdomen was disinfected with isodine, incised in the center, and laparotomy was performed after administration of anesthesia. The greater omentum and intestine were manually moved to expose the retroperitoneum after laparotomy. The retroperitoneum near the aorta and left kidney was bluntly dissected to form a pocket, and two fetal cloacas were transplanted into the pocket of each animal using a spatula. Thereafter, after transplantation, the pocket was closed with LIGACLIP (registered trademark). Next, three fetal cloacas were similarly transplanted into the greater omentum, and the pocket was closed with LIGACLIP (registered trademark). Thus, it was covered with greater omentum tissue so that the fetal cloaca would not fall off. After transplanting the fetal cloaca around the aorta and into the greater omentum, the abdomen and the wound were closed, and the surgery was completed. [Recovery of the greater omentum fetal kidney / fetal bladder around the aorta and anastomosis of the urinary tract] Thirty-three days after transplantation, the abdomen was opened again using a midline abdominal incision. After laparotomy, the greater omentum was exposed, and the entire kidney derived from the transplanted fetal cloaca was identified using the clipped portion with LIGACLIP (registered trademark) as a guide. Next, the kidney derived from the fetal cloaca was dissected together with the surrounding greater omentum tissue to avoid damage, and the tissue was recovered. Similarly, after the kidney derived from the fetal cloaca transplanted to the para-aortic portion was also identified, the regions around the left renal artery / vein and the left ureter were dissected, and the nearby abdominal aorta and inferior vena cava were exposed. After the left renal artery, the left renal vein and the left ureter were ligated, and the left kidney was removed. After removal, urinary reconstruction was performed through an end-to-end anastomosis of one of the recipient's fetal bladders and the left ureter by parachute suture. Finally, after urinary reconstruction, the abdomen and the wound were closed, and the surgery was completed. [Recovery of the para-aortic fetal kidney after ureter formation] At 50 or 79 days after transplantation, the abdomen was reopened by a midline abdominal incision. After laparotomy, the greater omentum and intestine were manually moved to identify the transplanted tissue around the aorta. Further, after identification, the right renal artery was ligated, and approximately 100 mL of extracellular fluid (normal saline, glucose, and VITAMEDIN (registered trademark)) was administered from the intravenous line together with an intravenous injection of furosemide (20 mg) and indigo carmine (20 mg). Observation was continued for 15 minutes or more, and urine production from the transplanted tissue was confirmed. Thereafter, the transplanted tissue was dissected together with the surrounding tissue, evaluated, and the experiment was terminated. [Immunosuppression, Anti - inflammation, and Supportive Therapy] The details of the immunosuppressive therapy are shown in Table 1. The start date is set with the transplantation date as zero (0), and before that is indicated by minus (-). Specifically, this includes tacrolimus (0.02 mg / kg im × 2 / day; start date - 9 days), abatacept (50 mg / kg iv × 1 / week; start date 7 days), and mycophenolate mofetil (40 mg / kg po × 2 / day; start date - 5 days). Methylprednisolone (10 mg / kg iv) was also administered from the start date 0 day, and switched to oral administration (5 mg / kg) the next day. The dosage was gradually reduced to 4, 3, 2, 1, 0.5, 0.25 mg / kg every 5 days, and then the administration of 0.25 mg / kg was maintained until autopsy. Furthermore, thymoglobulin (10 mg / kg iv on day 3), rituximab (10 mg / kg iv on day 2), tocilizumab (10 mg / kg sc on - 1, 7, and 14 days), and etanercept (0.5 mg / kg sc on 0, 3, 6, and 10 days) were administered. The concentrations of tacrolimus and mycophenolate mofetil in the blood were measured twice a week, and the dosage was appropriately adjusted based on the target trough level (the TAC trough value was 15 - 20 ng / mL from - 9 to 2 days and 9 - 12 ng / mL from 3 days on; the MMF trough value was 4 - 6 μg / mL). [Monitoring of Recipient Monkeys] The daily diet and activities of the recipient monkeys were checked, and support was provided by supplementation as necessary. Furthermore, the general condition of the monkeys was evaluated by measuring body weight, body temperature, SpO2, and blood sampling twice a week. In addition to TAC and MMF, RBC, Hb, Ht (MCV, MCH, and MCHC), RET, WBC (neutrophils, lymphocytes, monocytes, eosinophils, basophils), Plt, PT, APTT, Fbg, TP, Alb, T - Bil, LDH, AST, ALT, ALP, γGTP, CK, BUN, Cr, Na, K, Cl, Ca, P, Glu, T - Cho, TG, and CRP were also measured via blood tests. [Histological and Immunohistochemical Evaluation] The collected specimens were fixed with 4% paraformaldehyde and paraffin-embedded. Next, they were separated into 4-μm-thick sections and stained with hematoxylin and eosin (HE), periodic acid Schiff, and Masson's trichrome. For immunohistochemistry, 4-μm-thick serial sections from formalin-fixed specimens were deparaffinized and hydrated using ethanol in a stepwise concentration with respect to deionized water. The tissue sections for CD3 staining were treated in 0.01 M sodium citrate buffer using a pressure cooker for 10 seconds (the buffer was preheated for 30 seconds) for antigen retrieval. After antigen retrieval, all the sections were gently washed with PBS. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 20 minutes. After washing with PBS, all the sections were blocked with 5% skim milk for 30 minutes. Next, all the sections were incubated overnight at 4°C with an anti-CD3 antibody (MA5-12577, Invitrogen, Carlsbad, CA, USA). After washing with PBS, the sections were incubated with HISTOFINE SIMPLESTAIN (registered trademark) (424151, Nichirei, Japan) for 30 minutes. Diaminobenzidine was used as a chromogen, and hematoxylin was used as a counterstain. [Experiment 1: Immunological advantages of fetal kidney against heterologous rejection] The dosage of the immunosuppressive drug was increased or decreased based on the general condition and the results of blood biochemical examinations. The obtained pharmacokinetics are shown in FIGS. 1(A) and (B) (the results of Experiment 2 are also shown in FIGS. 1(C) and (D)). Also, after the start of immunosuppressant administration, a temporary increase in liver enzymes was observed in all cases, and the absorption of MMF was confirmed. Postoperative anemia was also observed. On the 7th day after surgery, the condition suddenly deteriorated, and one case (corresponding to Fig. 1(B)) in which neonatal pig kidneys and fetal cloacae were transplanted failed. Other cases in which neonatal pig kidneys and fetal cloacae were transplanted were relatively stable, but due to the deterioration of the condition and the migration of platelets, the abdomen was reopened on the 13th day after transplantation, and the neovascularized pig kidneys and the transplanted cloacae were simultaneously removed, and a biopsy of the cloacae was performed. The removed transplanted kidney was significantly swollen compared to that at the time of transplantation (weight 21 - 124 g, maximum diameter 5.2 - 10 cm), and the color also changed to dark red. On the other hand, the cloacae were visually preserved (see Figs. 2 and 3). In the transplanted kidney, according to pathological evaluation, the tissue was almost destroyed, and a large number of multinucleated cells deeply stained with HE staining and CD3-positive cells were observed (see Figs. 4(A)-(C)). On the other hand, the fetal kidneys (cloacae) recovered simultaneously with the transplanted kidney retained almost all tissue structures such as glomeruli and renal tubules. Furthermore, there was no infiltration of nuclear atypia and CD3-positive cells (see Figs. 4(D)-(F)). After closing the abdomen again, the same monkey continued to receive an immunosuppressant. After the transplanted pig kidney was removed, the monkey recovered and was relatively stable until the day of sacrifice 27 days after transplantation. The two recovered fetal kidneys are shown in Fig. 2. Although the tissue structure was preserved 13 days after transplantation, infiltration of CD3-positive cells was observed in part (see Figs. 4(G)-(I)). [Experiment 2: Growth of Fetal Cloacae with Administration of FDA-Approved Immunosuppressants] The urinary tract anastomosis between the bladder grown from the transplanted fetal cloacae and the recipient was performed on the 33rd day after about 4 weeks of tissue transplantation. The transplanted tissue was initially scheduled to be evaluated as a standard 6 weeks after the urinary tract anastomosis. However, after the anastomosis, the condition of one case gradually deteriorated, so it was euthanized after removing the transplanted tissue 50 days after transplantation. Since the other cases were relatively stable, the tissue was recovered 46 days after the anastomosis (79 days after transplantation) as scheduled. The major axis length of the largest fetal kidney recovered from the greater omentum 33 days after transplantation was approximately 800 μm (see Fig. 5(A)). The tissue structures such as glomeruli and renal tubules were mostly preserved as in Experiment 1, and no cell infiltration was observed. The space of the Bowman's capsule (also called glomerular capsule) was slightly enlarged based on visual inspection, indicating that urine was being produced and the kidney was functioning. The major axis length of the largest fetal kidney recovered from the para-aortic region 50 days after transplantation was approximately 1.5 mm. Furthermore, the major axis length of the largest fetal kidney recovered from the para-aortic region 79 days after transplantation was approximately 2 mm (see Figs. 5(B) and (C)). In the histopathological evaluation, no significant change was observed in the degree of cell infiltration in the fetal kidney at 33, 50, and 79 days after transplantation (see Fig. 6). This example is the first experimental result of directly comparing the immunogenicity of neonatal and fetal kidneys in xenotransplantation between non-genetically modified pigs and monkeys, successfully growing the fetal kidney (chlorocebus) in the long term after ureteral anastomosis, and observing the process. These experimental results confirmed that in xenotransplantation, the fetal kidney functions more favorably than the neonatal kidney, and long-term engraftment of the fetal kidney is possible. When transplanting the kidney of a mature pig into a monkey, long-term engraftment is difficult even in genetically modified pigs administered only FDA-approved immunosuppressive drugs. In the present invention, transplantation of the neonatal kidney of a pig deteriorated the condition of one recipient monkey. The strong rejection reaction against the transplanted kidney was consistent with the results of previous reports. However, inflammatory cell infiltration and tissue destruction characteristic of the rejection reaction in the neonatal pig kidney were hardly observed in the contemporaneous fetal kidney (chlorocebus renal primordium). In the case of the pig fetal kidney, if an appropriate period such as 4 weeks is selected, it has been established that the fetal kidney matures into kidney tissue that produces urine without differentiating into other tissues. Furthermore, the blood vessels of the xenogeneic recipient flow into the fetal kidney during the transplantation process, and the expression of donor antigens decreases during the developmental stage of the kidney itself. This suggests that the rejection reaction of chlorocebus is less likely to occur in xenotransplantation. However, in Experiment 1 in which neonatal and fetal kidneys were transplanted simultaneously, inflammatory cell infiltration was observed in the fetal kidney despite the shorter course compared to Experiment 2. Therefore, when there are elements that strongly induce an inflammatory response, such as in the kidneys of newborn piglets, inflammation may spread to the kidneys of the fetus, which are considered to have low immunogenicity. Furthermore, natural antibodies such as anti-pig IgE / A antibodies may be involved in the recipient pig's immune response, but they are thought not to have a significant impact on the development of the fetal kidneys. As confirmed in Experiment 2, long-term engraftment can be expected by reducing the type and dose of immunosuppressive drugs in the transplantation of only fetal kidneys (chloracal). To develop the fetal kidneys into more mature kidneys of the recipient, not only the control of rejection but also the establishment of the urinary excretion system is necessary, which was achieved by this example. In conclusion, in this example, the transplantation and long-term engraftment of fetal kidneys using a pig-to-pig preclinical model were successful without using genetically modified animals. This result is an important finding in developing chimeric fetal kidneys injected with human-derived renal progenitor cells to achieve humanized kidneys. According to the present invention, when transplanting to primates, it is possible to provide a transplant material and a kidney transplant kit that can suppress the recipient's rejection reaction and enable long-term engraftment.

Claims

1. A kidney transplantation kit comprising a transplantation material for renal xenotransplantation into primates, which contains a kidney primordium with a bladder not accompanied by blood vessels derived from porcine fetuses, and a combination of drugs used based on an immunosuppression protocol.

2. The kidney transplantation kit according to claim 1, wherein the kidney primordium with a bladder is derived from porcine fetuses at 4 to 6 weeks of gestation.

3. The kidney transplantation kit according to claim 1 or 2, wherein the kidney primordium with a bladder is derived from wild-type porcine fetuses.

4. The kidney transplantation kit according to claim 1 or 2, wherein the kidney primordium with a bladder further contains nephron progenitor cells derived from humans.

5. The immunosuppression protocol is (1) administering at least one immunosuppressant selected from thymoglobulin and anti-CD20 antibody, and (2) administering at least one immunosuppressant selected from a co-stimulation signal inhibitor via CD28, a calcineurin inhibitor, an inosine monophosphate dehydrogenase inhibitor, a steroid, an anti-CD25 antibody, and an anti-CD40 antibody. The kidney transplantation kit according to claim 1 or 2, which comprises the above.

6. The immunosuppression protocol further (3) administering at least one anti-inflammatory agent selected from an anti-IL-6 receptor antibody and a TNF-α inhibitor, and / or (4) administering at least one adjuvant selected from a platelet aggregation inhibitor, an anticoagulant, an antibacterial agent, a hematopoietic agent, a gastric acid secretion inhibitor, and an antiviral agent. The kidney transplantation kit according to claim 5, which comprises the above.

7. The immunosuppression protocol is (1) independently administering thymoglobulin and anti-CD20 antibody 2 to 3 days before transplantation, (2) administering a co-stimulation signal inhibitor via CD28 every 7 days after transplantation, or immediately after transplantation and every 7 days, (3) continuously administering a calcineurin inhibitor from 9 days before transplantation, (4) continuously administering an inosine monophosphate dehydrogenase inhibitor from 5 days before transplantation, (5) continuously administering a steroid from the day of transplantation, (6) administering an anti-IL-6 receptor antibody 1 to 3 times on the day before transplantation and approximately every 7 days after transplantation, (7) administering a TNF-α inhibitor every 3 to 4 days starting immediately after transplantation, and (8) administering at least one agent selected from a platelet aggregation inhibitor, an anticoagulant, an antibacterial agent, a hematopoietic agent, a gastric acid secretion inhibitor, and an antiviral agent as an adjuvant. The kidney transplantation kit according to claim 1 or 2, which comprises the above.

8. The immunosuppressive protocol is administering an anti-CD20 antibody about 2 to 3 weeks before transplantation; continuously administering an inosine monophosphate dehydrogenase inhibitor starting about 2 weeks before transplantation; administering an anti-CD25 antibody on the day of transplantation and on the fourth day; continuously administering a calcineurin inhibitor and a steroid starting about 1 week before transplantation; and administering at least one agent selected from a platelet aggregation inhibitor, an anticoagulant, an antibacterial agent, a hematopoietic agent, a gastric acid secretion inhibitor, and an antiviral agent as an adjuvant. The kidney transplantation kit according to claim 1 or 2, comprising the above.

9. A transplantation material for renal xenotransplantation into a primate, which comprises a kidney primordium with a bladder without porcine fetal-derived blood vessels and is used based on an immunosuppressive protocol, wherein the immunosuppressive protocol comprises administering at least one immunosuppressive agent selected from thymoglobulin and an anti-CD20 antibody; and administering at least one immunosuppressive agent selected from a co-stimulation signal inhibitor via CD28, a calcineurin inhibitor, an inosine monophosphate dehydrogenase inhibitor, a steroid, an anti-CD25 antibody, and an anti-CD40 antibody. The transplantation material comprising the above.

10. The transplantation material according to claim 9, wherein the immunosuppressive protocol further comprises administering at least one anti-inflammatory agent selected from an anti-IL-6 receptor antibody and a TNF-α inhibitor; and / or administering at least one adjuvant selected from a platelet aggregation inhibitor, an anticoagulant, an antibacterial agent, a hematopoietic agent, a gastric acid secretion inhibitor, and an antiviral agent. The transplantation material according to claim 9, comprising the above.

11. A transplantation material for renal xenotransplantation into a primate, which comprises a kidney primordium with a bladder without porcine fetal-derived blood vessels and is used based on an immunosuppressive protocol, wherein the immunosuppressive protocol comprises independently administering thymoglobulin and an anti-CD20 antibody 2 to 3 days before transplantation; administering a co-stimulation signal inhibitor via CD28 every 7 days after transplantation, or immediately after transplantation and every 7 days; continuously administering a calcineurin inhibitor starting 9 days before transplantation; continuously administering an inosine monophosphate dehydrogenase inhibitor starting 5 days before transplantation; continuously administering a steroid starting on the day of transplantation; Administering an anti-IL-6 receptor antibody 1 to 3 times on the day before transplantation and approximately every 7 days after transplantation; Administering a TNF-α inhibitor every 3 to 4 days starting immediately after transplantation; and Administering at least one agent selected from a platelet aggregation inhibitor, an anticoagulant, an antibacterial agent, a hematopoietic agent, a gastric acid secretion inhibitor, and an antiviral agent as an adjuvant; A transplant material comprising the same.

12. A transplant material used for renal xenotransplantation into a primate, which comprises a kidney primordium with a bladder without porcine fetal-derived blood vessels and is used based on an immunosuppressive protocol, wherein the immunosuppressive protocol is as follows: Administering an anti-CD20 antibody approximately 2 to 3 weeks before transplantation; Continuously administering an inosine monophosphate dehydrogenase inhibitor starting approximately 2 weeks before transplantation; Administering an anti-CD25 antibody on the day of transplantation and on the 4th day; Continuously administering a calcineurin inhibitor and a steroid starting approximately 1 week before transplantation; and Administering at least one agent selected from a platelet aggregation inhibitor, an anticoagulant, an antibacterial agent, a hematopoietic agent, a gastric acid secretion inhibitor, and an antiviral agent as an adjuvant; A transplant material comprising the same.

13. The transplant material according to any one of claims 9 to 12, wherein the kidney primordium with a bladder is derived from a porcine fetus at 4 to 6 weeks of gestation.

14. The transplant material according to claim 13, wherein the kidney primordium with a bladder is derived from a wild-type porcine fetus.

15. The transplant material according to claim 13, wherein the kidney primordium with a bladder further comprises human-derived nephron progenitor cells.