Long-term viable chimeric nephrons generated from progenitor cells are a reliable model in cisplatin-induced toxicity.

Kidney organoids have shown promise as evaluation tools, but their in vitro maturity remains limited. Transplantation into adult mice has aided in maturation; however, their lack of urinary tract connection limits long-term viability. Thus, long-term viable generated nephrons have not been demonstrated. In this study, we present an approachable method in which mouse and rat renal progenitor cells are injected into the developing kidneys of neonatal mice, resulting in the generation of chimeric nephrons integrated with the host urinary tracts. These chimeric nephrons exhibit similar maturation to the host nephrons, long-term viability with excretion and reabsorption functions, and cisplatin-induced renal injury in both acute and chronic phases, as confirmed by single-cell RNA-sequencing. Additionally, induced human nephron progenitor cells differentiate into nephrons within the neonatal kidneys. Collectively, neonatal injection represents a promising approach for in vivo nephron generation, with potential applications in kidney regeneration, drug screening, and pathological analysis.

Evaluation of the ability of human induced nephron progenitor cells to form chimeric renal organoids using mouse embryonic renal progenitor cells.

The number of patients with end-stage renal failure is increasing annually worldwide and the problem is compounded by a shortage of renal transplantation donors. In our previous research, we have shown that transplantation of renal progenitor cells into the nephrogenic region of heterologous fetuses can induce the development of nephrons. We have also developed transgenic mice in which specific renal progenitor cells can be removed by drugs. By combining these two technologies, we have succeeded in generating human-mouse chimeric kidneys in fetal mice. We hope to apply these technologies to regenerative medicine. The quality of nephron progenitor cells (NPCs) derived from human pluripotent stem cells is important for the generation of chimeric kidneys, but there is currently no simple evaluation system for the chimerogenic potential of human NPCs. In this study, we focused on the fact that the re-aggregation of mouse renal progenitor cells can be used for nephron formation, even when merged into single cells. First, we examined the conditions under which nephron formation is likely to occur in mice during re-aggregation. Next, to improve the differentiation potential of human NPCs derived from pluripotent stem cells, NPCs were sorted using Integrin subunit alpha 8 (ITGA8). Finally, we demonstrated chimera formation between different species by mixing mouse cells with purified, selectively-induced human NPCs under optimum conditions. We observed these chimeric organoids at different time points to learn about these human-mouse chimeric structures at various stages of renal development. We found that the rate of chimera formation was affected by the purity of the human NPCs and the cell ratios used. We demonstrated that chimeric nephrons can be generated using a simple model, even between distant species. We believe that this admixture of human and mouse renal progenitor cells is a promising technology with potential application for the evaluation of the chimera formation abilities of NPCs.

Possible Use of Non-purine Selective Xanthine Oxidoreductase Inhibitors for Prevention of Exercise-induced Acute Kidney Injury Associated with Renal Hypouricemia.

Exercise-induced acute kidney injury (EIAKI) is frequently complicated with renal hypouricemia (RHUC). In patients with RHUC, limiting anaerobic exercise can prevent EIAKI. However, it is challenging to reduce exercise intensity in athletes. We herein report a 16-year-old Japanese football player with familial RHUC with compound heterozygous mutations in urate transporter 1 (URAT1) who presented with recurrent EIAKI. As prophylaxis (hydration during exercise) could not prevent EIAKI, febuxostat was initiated. EIAKI was not observed for 16 months despite exercising intensively. Hence, non-purine-selective xanthine oxidoreductase inhibitors may decrease the incidence of EIAKI in athletes with RHUC.

Generation of functional chimeric kidney containing exogenous progenitor-derived stroma and nephron via a conditional empty niche.

Generation of new kidneys can be useful in various research fields, including organ transplantation. However, generating renal stroma, an important component tissue for structural support, endocrine function, and kidney development, remains difficult. Organ generation using an animal developmental niche can provide an appropriate in vivo environment for renal stroma differentiation. Here, we generate rat renal stroma with endocrine capacity by removing mouse stromal progenitor cells (SPCs) from the host developmental niche and transplanting rat SPCs. Furthermore, we develop a method to replace both nephron progenitor cells (NPCs) and SPCs, called the interspecies dual replacement of the progenitor (i-DROP) system, and successfully generate functional chimeric kidneys containing rat nephrons and stroma. This method can generate renal tissue from progenitors and reduce xenotransplant rejection. Moreover, it is a safe method, as donor cells do not stray into nontarget organs, thus accelerating research on stem cells, chimeras, and xenotransplantation.

In vivo regeneration of neo-nephrons in rodents by renal progenitor cell transplantation.

Renal progenitor cells induced from pluripotent stem cells have attracted attention as a cell source for organ regeneration. Here, we report an in vivo protocol for the regeneration of urine-producing nephrons, i.e., neo-nephrons, in mice. We outline steps to transplant exogenous renal progenitor cells into the nephrogenic zone of transgenic mice and subsequently analyze these neo-nephrons. For complete details on the use and execution of this protocol, please refer to Fujimoto et al. (2020).

Techniques of orthotopic renal transplantation. II. Size-matched porcine grafts in monkey recipients

ABSTRACT Purpose As a classical xenotransplantation model, porcine kidneys have been transplanted into the lower abdomen of non-human primates. However, we have improved upon this model by using size-matched grafting in the orthotopic position. The beneficial aspects and surgical details of our method are reported herein. Methods Donors were two newborn pigs (weighting 5 to 6 kg) and recipients were two cynomolgus monkeys (weighting, approximately, 7 kg). After bilateral nephrectomy, kidneys were cold-transported in Euro-Collins solution. The porcine kidney was transplanted to the site of a left nephrectomy and fixed to the peritoneum. Results Kidneys transplanted to the lower abdomen by the conventional method were more susceptible to torsion of the renal vein (two cases). In contrast, early-stage blood flow insufficiency did not occur in orthotopic transplants of theleft kidney. Conclusions Size-matched porcine-primate renal grafting using our method of transplanting tothe natural position of the kidneys contributes to stable post-transplant blood flow to the kidney.

Generation of Human Renal Vesicles in Mouse Organ Niche Using Nephron Progenitor Cell Replacement System.

Animal fetuses may be used for the regeneration of human organs. We have previously generated a transgenic mouse model that allows diphtheria toxin (DT)-induced ablation of Six2-positive nephron progenitor cells (NPCs). Elimination of existing native host NPCs enables their replacement with donor NPCs, which can generate neo-nephrons. However, this system cannot be applied to human NPCs, because DT induces apoptosis in human cells. Therefore, the present study presents a transgenic mouse model for the ablation of NPCs using tamoxifen, which does not affect human cells. Using this system, we successfully regenerate interspecies neo-nephrons, which exhibit urine-producing abilities, from transplanted rat NPCs in a mouse host. Transplantation of human induced pluripotent stem cell (iPSC)-derived NPCs results in differentiation into renal vesicles, which connect to the ureteric bud of the host. Thus, we demonstrate the possibility of the regeneration of human kidneys derived from human iPSC-derived NPCs via NPC replacement.

Mesangial cell regeneration from exogenous stromal progenitor by utilizing embryonic kidney.

Kidney regenerative medicine is expected to be the solution to the shortage of organs for transplantation. In a previous report, we transplanted exogenous renal progenitor cells (RPCs) including nephron progenitor cells (NPCs), stromal progenitor cells (SPCs), and the ureteric bud (UB) into the nephrogenic zone of animal embryos and succeeded in regenerating new nephrons from exogenous NPCs through a fetal developmental program. However, it was unknown whether the renal stromal lineage cells were regenerated from SPCs. The present study aimed to verify the differentiation of SPCs into mesangial cells and renal stromal lineage cells. Here, we found that simply transplanting RPCs, including SPCs, into the nephrogenic zone of wild-type fetal mice was insufficient for differentiation of SPCs. Therefore, to enrich the purity of SPCs, we sorted cells from RPCs by targeting platelet-derived growth factor receptor alpha (PDGFRa) which is a cell surface marker for immature stromal cells and transplanted the PDGFRa-positive sorted cells. As a result, we succeeded in regenerating a large number of mesangial cells and other renal stromal lineage cells including interstitial fibroblasts, vascular pericytes, and juxtaglomerular cells. We have established the method for regeneration of stromal cells from exogenous SPCs that may contribute to various fields, such as regenerative medicine and kidney embryology, and the creation of disease models for renal stromal disorders.

Kidney Regeneration in Later-Stage Mouse Embryos via Transplanted Renal Progenitor Cells.

BACKGROUND: The limited availability of donor kidneys for transplantation has spurred interest in investigating alternative strategies, such as regenerating organs from stem cells transplanted into animal embryos. However, there is no known method for transplanting cells into later-stage embryos, which may be the most suitable host stages for organogenesis, particularly into regions useful for kidney regeneration. METHODS: We demonstrated accurate transplantation of renal progenitor cells expressing green fluorescent protein to the fetal kidney development area by incising the opaque uterine muscle layer but not the transparent amniotic membrane. We allowed renal progenitor cell-transplanted fetuses to develop for 6 days postoperatively before removal for analysis. We also transplanted renal progenitor cells into conditional kidney-deficient mouse embryos. We determined growth and differentiation of transplanted cells in all cases. RESULTS: Renal progenitor cell transplantation into the retroperitoneal cavity of fetuses at E13-E14 produced transplant-derived, vascularized glomeruli with filtration function and did not affect fetal growth or survival. Cells transplanted to the nephrogenic zone produced a chimera in the cap mesenchyme of donor and host nephron progenitor cells. Renal progenitor cells transplanted to conditional kidney-deficient fetuses induced the formation of a new nephron in the fetus that is connected to the host ureteric bud. CONCLUSIONS: We developed a cell transplantation method for midstage to late-stage fetuses. In vivo kidney regeneration from renal progenitor cells using the renal developmental environment of the fetus shows promise. Our findings suggest that fetal transplantation methods may contribute to organ regeneration and developmental research.

In vivo regeneration of interspecies chimeric kidneys using a nephron progenitor cell replacement system.

Kidney regeneration is expected to be a new alternative treatment to the currently limited treatments for chronic kidney disease. By transplanting exogeneous nephron progenitor cells (NPCs) into the metanephric mesenchyme of a xenogeneic foetus, we aimed to regenerate neo-kidneys that originate from transplanted NPCs. Previously, we generated a transgenic mouse model enabling drug-induced ablation of NPCs (the Six2-iDTR mouse). We demonstrated that eliminating existing native host NPCs allowed their 100% replacement with donor mouse or rat NPCs, which could generate neo-nephrons on a culture dish. To apply this method to humans in the future, we examined the possibility of the in vivo regeneration of nephrons between different species via NPC replacement. We injected NPCs-containing rat renal progenitor cells and diphtheria toxin below the renal capsule of E13.5 metanephroi (MNs) of Six2-iDTR mice; the injected MNs were then transplanted into recipient rats treated with immunosuppressants. Consequently, we successfully regenerated rat/mouse chimeric kidneys in recipient rats receiving the optimal immunosuppressive therapy. We revealed a functional connection between the neo-glomeruli and host vessels and proper neo-glomeruli filtration. In conclusion, we successfully regenerated interspecies kidneys in vivo that acquired a vascular system. This novel strategy may represent an effective method for human kidney regeneration.

Regenerative potential of induced pluripotent stem cells derived from patients undergoing haemodialysis in kidney regeneration.

Kidney regeneration from pluripotent stem cells is receiving a lot of attention because limited treatments are currently available for chronic kidney disease (CKD). It has been shown that uremic state in CKD is toxic to somatic stem/progenitor cells, such as endothelial progenitor and mesenchymal stem cells, affecting their differentiation and angiogenic potential. Recent studies reported that specific abnormalities caused by the non-inherited disease are often retained in induced pluripotent stem cell (iPSC)-derived products obtained from patients. Thus, it is indispensable to first assess whether iPSCs derived from patients with CKD due to non-inherited disease (CKD-iPSCs) have the ability to generate kidneys. In this study, we generated iPSCs from patients undergoing haemodialysis due to diabetes nephropathy and glomerulonephritis (HD-iPSCs) as representatives of CKD-iPSCs or from healthy controls (HC-iPSCs). HD-iPSCs differentiated into nephron progenitor cells (NPCs) with similar efficiency to HC-iPSCs. Additionally, HD-iPSC-derived NPCs expressed comparable levels of NPC markers and differentiated into vascularised glomeruli upon transplantation into mice, as HC-iPSC-derived NPCs. Our results indicate the potential of HD-iPSCs as a feasible cell source for kidney regeneration. This is the first study paving the way for CKD patient-stem cell-derived kidney regeneration, emphasising the potential of CKD-iPSCs.

Optimal route of diphtheria toxin administration to eliminate native nephron progenitor cells in vivo for kidney regeneration.

To address the lack of organs for transplantation, we previously developed a method for organ regeneration in which nephron progenitor cell (NPC) replacement is performed via the diphtheria toxin receptor (DTR) system. In transgenic mice with NPC-specific expression of DTR, NPCs were eliminated by DT and replaced with NPCs lacking the DTR with the ability to differentiate into nephrons. However, this method has only been verified in vitro. For applications to natural models, such as animal fetuses, it is necessary to determine the optimal administration route and dose of DT. In this study, two DT administration routes (intra-peritoneal and intra-amniotic injection) were evaluated in fetal mice. The fetus was delivered by caesarean section at E18.5, and the fetal mouse kidney and RNA expression were evaluated. Additionally, the effect of the DT dose (25, 5, 0.5, and 0.05 ng/fetus-body) was studied. Intra-amniotic injection of DT led to a reduction in kidney volume, loss of glomeruli, and decreased differentiation marker expression. The intra-peritoneal route was not sufficient for NPC elimination. By establishing that intra-amniotic injection is the optimal administration route for DT, these results will facilitate studies of kidney regeneration in vivo. In addition, this method might be useful for analysis of kidney development at various time points by deleting NPCs during development.

A refractory case of subclinical antibody-mediated rejection due to anti-HLA-DQ antibody in a kidney transplant patient.

We herein report a refractory case of subclinical antibody-mediated rejection (AMR) due to anti-HLA-DQ antibody in a kidney transplant patient. A 45-year-old man was admitted for a protocol biopsy; he had a serum creatinine (S-Cr) level of 1.8 mg/dL 3 years following primary kidney transplantation. Histological examination revealed moderate to severe inflammatory cell infiltration in the peritubular capillaries. Thorough laboratory examination showed that the patient had donor-specific antibodies (DSAbs) to DR9 and DQ9. Considering both the histological and laboratory findings, we diagnosed acute antibody-mediated rejection. The patient underwent 3 days of consecutive steroid pulse therapy, intravenous immunoglobulin (IVIG), and plasma exchange. We also administered rituximab (200 mg/body). Six months after the treatment, a second allograft biopsy revealed the progression of interstitial fibrosis and tubular atrophy and persistence of mild peritubular capillaritis. Further analysis showed that the anti-DR9 antibodies had disappeared, but that the mean fluorescence intensity value of the anti-DQ9 antibodies had increased. Therefore, we repeated the plasma exchange and IVIG. Allograft function was stable throughout the course of treatment, and the S-Cr level remained at 1.8 mg/dL. This case report demonstrates the difficulty of treating AMR due to the presence of anti-DQ DSAbs and the necessity for subsequent therapies in refractory cases.