Fetal Kidney Transplantation into In Utero Fetuses

doi:10.21203/rs.3.rs-4878169/v1

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Fetal Kidney Transplantation into In Utero Fetuses

https://doi.org/10.21203/rs.3.rs-4878169/v1

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Potter sequence consists of various symptoms associated with renal dysplasia. For bilateral renal agenesis, there is no hope of survival. As a novel therapeutic approach for Potter sequence, we developed a unique approach of “transplantation of fetal kidneys from a different species during the fetal period.” In this study, we first validated the approach using allogeneic transplantation. Fetal kidneys with bladders from green fluorescent protein-expressing rats (embryonic day 14.0–16.5) were subcutaneously transplanted into allogeneic rat fetuses in utero (embryonic day 18.0–18.5). After birth, the transplanted fetal kidneys were confirmed to have urine production capability. Furthermore, long-term (up to 150 days) urine production was sustained. Next, we performed xenotransplantation. The transplantation of mouse fetal kidneys into rat fetuses in utero led to the maturation of renal tissue structures. We demonstrated organ transplantation into in utero fetuses using fetal kidneys as donor organs for fetal therapy.

Health sciences/Nephrology/Renal replacement therapy/Continuous renal replacement therapy

Health sciences/Medical research/Preclinical research

Potter sequence, which is characterized by bilateral renal dysplasia, oligohydramnios, and consequent pulmonary hypoplasia, is one of the most serious perinatal diseases ^1,2,3,4. Bilateral renal dysplasia resulting in Potter sequence occurs in 1 in 4,000 individuals ^5,6. In cases with bilateral renal agenesis, either fetal death occurs or the affected infants die within 12 hours after birth due to pulmonary hypoplasia in all cases ⁶. In other words, without intervention, the neonatal mortality rate is 100% ⁷. Even if pulmonary hypoplasia is prevented through continuous amniotic fluid infusion therapy and immediate postnatal death is avoided, dialysis therapy is essential from birth. However, because of their prematurity and associated abdominal complications, these infants often fail to reach a stage at which dialysis can be safely initiated and sustained, leading to an exceedingly high mortality rate from this condition ⁸. Therefore, there is hopeful anticipation that interventions serving as a bridge to achieve a state where dialysis can be safely performed will markedly improve life expectancy. Currently, there is no established effective treatment for such severe fetal renal failure, and dialysis therapy is performed as a limited treatment option after birth.

We have previously reported that rat fetal kidneys can grow, differentiate, and produce urine in the body (retroperitoneal space) of adult rats ^9–13. Similar phenomena have been confirmed in interspecies combinations, such as rat–mouse and pig–mouse pairs ^14,15. We have developed a method of transplantation of the fetal kidney, ureter, and bladder unit as an approach to avoid hydronephrosis for a limited period ⁹. This urological unit is called fetal kidneys with bladders (metanephroi with bladders: MNBs).

Fetal kidneys at this stage of development lack the necessary glomerular structure with vascular invasion for use as donor organs in our studies ^16,17. After transplantation, recipient-derived blood vessels invade the transplanted fetal kidneys and form glomeruli. The vascular endothelium is at the forefront of rejection in organ transplantation, and the advantage of constructing the vascular endothelium of the transplanted organ using self-vessels is that these vessels are less likely to be the target of rejection ^18,19. For fetal treatment, the current kidney transplantation with vascular anastomosis does not fit the organ size and seems impossible as a fetal surgical technique. In contrast, fetal kidneys are appropriately sized and can be transplanted without vascular anastomosis. Therefore, we considered transplanting xenogeneic fetal kidneys with bladders into intrauterine fetuses as a bridge therapy until dialysis can be stably performed. However, it is unclear whether MNBs can be transplanted, engrafted, and mature in the fetus that is targeted for treatment, with no such reported cases being available in the literature. In this study, we transplanted rat MNBs into the subcutaneous tissue of rat fetuses and confirmed their engraftment, maturation, and urine production. We also investigated the immunological advantages of using fetuses as recipients at the developmental stage of the immune system. In addition, the model of xenogeneic transplantation was validated by transplanting mouse fetal kidneys into rat fetuses, which confirmed the maturation of the transplanted kidneys and yielded less tissue damage caused by rejection compared to the transplantation of mouse fetal kidneys into adult rats.

We aimed to develop a novel treatment for fetuses with bilateral renal agenesis, a condition currently without treatment, and have conceived a concept of fetal therapy—the transplantation of xenogeneic fetal kidneys with bladders during the fetal period. This study demonstrates the effectiveness of organ transplantation during the fetal period and is expected to contribute to the development of groundbreaking treatments for congenital kidney diseases, such as the Potter sequence. Although there have been reports of the transplantation of smaller cells into the amniotic fluid ²⁰, peritoneal cavity ^{21, 22}, or retroperitoneal space ²³ of intrauterine fetuses, to the best of our knowledge, there have been no reports of the transplantation of larger tissue bodies, such as organs, into intrauterine fetuses accompanied by the endowment of function, including in rodent models. In this study, we successfully transplanted fetal organs during the fetal period and conducted an analysis of the transplanted tissue.

Transplantation of green fluorescent protein-Sprague–Dawley (GFP-SD) rat MNBs into the fetal subcutaneous space of SD rats

We developed a method for transplanting MNBs from GFP-SD rats into the subcutaneous space of rat fetuses in utero. An overview of the experiment is provided in Fig. 1a. One MNB extracted from a GFP-SD rat (embryonic day 14.0–16.5 (E14.0–16.5)) (Fig. 1b) was loaded onto the tip of a 15-16G needle. Pregnant SD rats (E18.0–18.5) were laparotomized through a midline incision, and the uterus was gently exteriorized from the abdominal cavity. The position of the fetuses was confirmed through the semitransparent uterine wall (Fig. 1c). The uterine wall was punctured with the 15-16G needle loaded with GFP rat MNBs on the dorsal side of the fetus, and the needle was further inserted into the fetal subcutaneous space while being monitored under a stereomicroscope (Fig. 1d, e; see Supplementary Movie 1 online). Approximately 0.1–0.5 mL of Hank’s balanced salt solution (HBSS) was ejected to transplant the GFP rat MNBs loaded onto the needle tip into the fetal subcutaneous space. Immediately after transplantation, the presence of GFP-positive tissue was confirmed using a fluorescence stereomicroscope to ensure proper transplantation (Fig. 1f).

To minimize the duration of surgery, 2–4 fetuses per mother were used as recipients (approximately 40 min of total anesthesia time was required for transplantation into the 4 fetuses). The uterus was gently returned to the abdominal cavity, and the incision was closed. Four days after transplantation, on E22, natural delivery occurred, and live offspring were successfully obtained. By puncturing the subcutaneous space of the fetuses in utero, the invasiveness of the procedure was reduced, and the fetuses survived the invasiveness of the thick 15-16G needle. The survival rate through natural delivery after transplantation surgery was 76%. On the day of natural delivery, the backs of all offspring were photographed using a fluorescence stereomicroscope to confirm the presence of GFP-positive tissue. Among the transplanted fetuses, GFP-positive tissue was confirmed in offspring with an average transplant success rate of 88% (Table 1).

Table 1

Transplant success rate.
	Number of live pups/ all fetuses	Survival rate [%]	Number of transplanted fetuses	Number of GFP-positive neonates	Success rate (%)
Case1	12/14	86	2	1	50
Case2	9/11	82	4	4	100
Case3	11/14	79	2	2	100
Case4	8/14	57	1	1	100
Average	−	76	−	−	88

Maturation of transplanted GFP-SD rat MNBs in neonates

After natural delivery on embryonic day 22, 4 days after the transplantation of the GFP-SD rat MNBs, the neonates were nursed by their biological mothers and grew. There was no significant difference in body weight gain between the transplanted (n = 3) and nontransplanted (n = 3) neonates, indicating the absence of growth impairment associated with the transplantation (Fig. 2a). GFP-positive tissue was observed on the body surface of the neonates using a fluorescence stereomicroscope up to 28 days posttransplantation (24 days after birth), and a gradual increase in the size of the transplanted MNBs was noted (Fig. 2b, c). On day 28 posttransplantation, the dorsal epidermis was incised, and the transplanted MNBs were examined, confirming the invasion of recipient subcutaneous blood vessels into the transplanted MNBs (Fig. 2d; Supplementary Fig. 1 online for the structure of the fetal kidney before transplantation). Percutaneous ultrasonography revealed the presence of a urinary cyst (Fig. 2e), suggesting that the transplanted MNBs were producing urine. Histologically, glomeruli containing nephrin-positive cells, lotus tetragonolobus lectin (LTL)-positive proximal tubules, and E-cadherin (ECAD)-positive distal tubules were observed, confirming the maturation of the MNBs (Fig. 2f, g). In contrast, when GFP-SD rat MNBs were transplanted into adult SD rats and the tissue was retrieved 14 days posttransplantation, rejection was observed and consequently, glomerular and tubular structures could not be identified (Fig. 2h). Compared with transplantation into adults, MNB transplantation into fetuses yielded a reduced rate of rejection, suggesting that MNB transplantation into fetuses has immunological advantages regarding GFP antigen and allogeneic grafts. Moreover, GFP-negative CD31-positive vascular endothelial cells were detected, suggesting that recipient-derived blood vessels invaded the transplanted structures and formed glomeruli, producing urine (Fig. 2i, j). Conversely, GFP-positive CD31-positive blood vessels were also observed, indicating the presence of donor-derived vasculature (Fig. 2k). These findings demonstrated the successful creation of a mature exogenous kidney by transplanting allogeneic MNBs into rat fetuses in utero.

Development of the urine excretion method for GFP-SD rat MNBs transplanted into the subcutaneous space

GFP-SD rat MNBs were transplanted into SD rat fetuses. Four days after transplantation, on embryonic day 22, natural delivery occurred. At 22 days posttransplantation (18 days after birth), a clearly raised structure, presumably the transplanted MNBs, was observed from the body surface (Fig. 3a). Percutaneous ultrasonography revealed a distinct accumulation of fluid with a long diameter of approximately 1 cm, indicating a sufficient urine production capacity for safe percutaneous aspiration (Fig. 3b). Subsequently, a single puncture was performed on the back of the animals using a 23-29G needle, and urine was aspirated (Fig. 3c; see Supplementary Movie 2 online). Thereafter, single punctures were repeated 1–2 times per week, and continuous and stable urine excretion to the outside of the body was successfully maintained up to 150 days posttransplantation (146 days after birth) (Fig. 3d). On average, it was shown that approximately 1 mL of urine was produced per day. In addition, the measurement of creatinine clearance (CCr) showed that it ranged from approximately 40 to 80 µL/min, confirming that the produced fluid was urine with solute-removal capacity (Fig. 3d). After confirming long-term urine excretion, tissue retrieval was performed at 150 days posttransplantation (146 days after birth) (Fig. 3e,f). Compared with the tissue image obtained at 28 days posttransplantation (Fig. 2f), a more extensive mature tubular structure was observed (Fig. 3g). Furthermore, Masson’s trichrome staining revealed a lack of fibrotic areas compared with tissue retrieval performed at 78 days posttransplantation without any aspiration punctures (Fig. 3h).

Therefore, it was confirmed that administering repeated aspiration punctures for urine excretion prevented hydronephrosis and allowed longer-term maturation of the MNBs. To investigate whether the frequency of 1–2 punctures per week was appropriate for optimal urine production, the correlation between the urine accumulation time between punctures (in this study, the minimum accumulation time between punctures was 24 h) and the amount of urine produced per 24 h was examined. It was found that shorter accumulation times tended to result in a higher urine output per 24 h (Fig. 3i).

To ensure reproducibility, similar MNB transplantation experiments into fetuses were performed 17 times. However, in allogeneic transplantation, only 23.5% (4/17) of the individuals developed a urinary cyst of sufficient size (a major diameter of at least 1 cm on ultrasonography), as described above. In all experiments, tissue retrieval was performed more than 3 weeks after transplantation, and it was inferred that rejection occurred to varying degrees in individuals with poor urine production. This suggests that fetal transplantation exhibits varying degrees of immunological advantage acquisition.

Verification of the immunological advantages of fetal transplantation

Using fetuses with developing immune systems as recipients may have immunological advantages. To investigate one possible underlying mechanism, the following experiment was conducted. Fetal kidneys were individually extracted from GFP-SD rats (E14.0–14.5). One fetal kidney was transplanted into an SD rat fetus in utero (E18.0–18.5). These SD strains have been maintained in an out bred condition. The other fetal kidney from the same individual was then cryopreserved using a previously reported protocol ²⁴. Four days after transplantation, on embryonic day 22, natural delivery occurred, and the pups were nursed by their mothers. One month later, the pups were weaned, and at 9 weeks after birth, when the immune system was fully mature, the cryopreserved fetal kidney was thawed and retransplanted into the retroperitoneal cavity of each individual, respectively. Tissue retrieval was performed 2 weeks after transplantation (i.e., 11 weeks after birth) (Fig. 4a). In the tissue, the GFP-positive areas were sufficiently preserved, and recipient-derived blood vessels invading the fetal kidneys were observed (Fig. 4b, c). Histologically, minimal lymphocyte infiltration was observed, and the glomerular and tubular structures were confirmed (Fig. 4d, e, f). Compared with the rejection observed in the tissue that was retrieved 2 weeks after the transplantation of GFP-SD rat MNBs into adult SD rats (at 8 weeks of age) (Fig. 2h), in which glomerular and tubular structures could not be identified, the rejection reaction was weak. Although further research is necessary to elucidate the immunological advantages of transplantation into fetuses, these results may help in understanding the underlying mechanisms.

Verification of xenotransplantation (mice to rats)

A similar experiment was conducted using xenotransplantation of GFP-B6 (C57BL/6) mouse fetal kidneys into SD rat fetuses. Four days after transplantation, on embryonic day 22, natural delivery occurred, and the pups were nursed by their mothers and grew. Tissue retrieval was performed after transplantation. Even without immunosuppressant administration, in the case of the transplantation into SD rat fetuses, the GFP-positive areas persisted 10 days after transplantation, and numerous CD3-positive cells infiltrated the tubular interstitial region; however, glomerular structures were observed (Fig. 5a, b). However, 18 days after transplantation, the GFP-positive areas were decreased, severe inflammatory cell infiltration was observed, and no glomerular structures were detected (Fig. 5c). In contrast, when GFP-B6 mouse fetal kidneys were transplanted into adult SD rats at 8 weeks of age and tissue was retrieved 3 days after transplantation, severe rejection was observed before maturation (Fig. 5d), suggesting that rejection is somewhat reduced even in xenotransplantation into fetuses. To reduce rejection, tacrolimus administration was performed. Furthermore, to transfer tacrolimus to the fetuses via the placenta, tacrolimus was subcutaneously administered to the mother rats at 0.2 mg/kg/day (every other day) after transplantation, and, after birth, the neonates themselves were subcutaneously administered tacrolimus at 0.1 mg/kg/day (every other day). In a preliminary evaluation, the placental transfer rate of tacrolimus from the mother rat to the fetuses was approximately 40% (see Supplementary Table 1). In the presence of tacrolimus administration, the GFP-positive areas were sufficiently preserved even at 18 days after transplantation (Fig. 5e); histologically, glomerular and tubular structures were observed (Fig. 5f). Immunostaining revealed the presence of ECAD-positive distal tubules (Fig. 5g). The CD31-positive vessels observed inside the GFP-positive glomerular structures were GFP-negative, indicating that the glomeruli were completely composed of the recipient (mouse)-derived vessels (Fig. 5h). Conversely, marked infiltration of CD3-positive cells was observed in the tubular interstitial region, confirming that tacrolimus monotherapy was insufficient to suppress rejection in the context of xenotransplantation (Fig. 5h).

In this study, we successfully transplanted urological units, i.e., MNBs consisting of a kidney, ureter, and bladder, into rat fetuses in utero using a needle via a transuterine approach. We obtained live offspring through natural delivery and created a mature exogenous kidney.

We focused on subcutaneous transplantation as the transplantation site. In previous studies using adult recipients in rodents, the retroperitoneal space was generally selected as the transplantation site. However, transplantation into the retroperitoneal space requires a laparotomy, which renders it unrealistic to perform transuterine transplantation. Interestingly, we discovered that subcutaneous transplantation ²⁵, which was considered disadvantageous in adult transplantation, was suitable as a site for fetal organs during fetal transplantation. We have previously attempted to transplant fetal kidneys into the subcutaneous space of adults ²⁵; however, in all cases, the development was poor, and no significant urine production was obtained. Subcutaneous transplantation had a low incidence of bleeding complications, and the fetal survival rate was high, at 76%. Expanding the transplantation bed as the fetus grows may also alleviate physical constraints. In addition to the physical and spatial advantages, we hypothesized that there might be specific advantages of the fetal subcutaneous space, as there was a remarkable difference in the development of the transplanted MNBs after transplantation, with urine production, compared to adult recipient subcutaneous transplantation.

It has been reported that the composition of the extracellular matrix (ECM) differs between the subcutaneous space of adults and fetuses ^{26, 27}. The ECM promotes the development of angiogenesis in kidney organoids that mimic embryonic organs ²⁸. In addition, some components of the ECM have been reported to play a role in directing the migration of endothelial cells, contributing to angiogenesis ²⁹. Thus, an analysis focusing on the unique ECM composition of the fetal subcutaneous space is necessary to understand the difference between adult and fetal subcutaneous transplantation.

The large amount of urine produced for such a long period by a single MNB recorded here has not been observed in retroperitoneal transplantation in adults. We also consider that the ease of urine excretion management through subcutaneous transplantation was an important advantage, as regular urine excretion is crucial for preventing hydronephrosis in the setting of fetal kidney transplantation. Once urine is produced by the fetal kidneys, if left untreated, the transplanted MNBs will develop hydronephrosis because of the lack of a urine excretion pathway, and eventually, renal function will be lost. Therefore, the formation of a urine excretion pathway becomes necessary at 3–4 weeks after transplantation, when urine production is observed. In previous studies, MNBs were transplanted into the retroperitoneal space of adults, making it difficult to excrete urine through percutaneous aspiration from the body surface. Therefore, a surgical method was employed to connect the urinary cyst to the host’s ureter by laparotomy ⁹. In our model, we took advantage of the fact that the transplanted MNBs were located subcutaneously and in close proximity to the body surface. We successfully performed safer and continuous percutaneous aspiration for 150 days, thus avoiding hydronephrosis and achieving urine excretion to the outside of the body.

After urine aspiration, it reaccumulates in the bladder over a certain period, but the optimal frequency of aspiration punctures for better urine production was unknown. Therefore, we investigated the appropriate frequency for administering aspiration punctures and found that shorter accumulation times between punctures resulted in larger urine production. This suggests that more continuous urine excretion, rather than intermittent excretion, may result in a higher urine output per unit of time. Considering safety, we performed aspiration punctures 1–2 times per week in this study; however, in the future, we aim to establish a method for continuous drainage, such as placing a catheter.

Moreover, when considering long-term engraftment, it is important to factors in the immunological advantages, especially in this rodent system. In the case of the allogeneic transplantation of MNBs into fetuses (SD rat–SD rat), the rejection was reduced to such an extent that urine production persisted for 150 days without the use of immunosuppressants. This may be attributed to two advantages of fetal kidney transplantation into fetuses in utero. One is that the donor organ used here was a fetal kidney, as previously reported ^{30, 31}, and the other is that the recipient was a fetus, a characteristic specific to this study. Regarding the former, it has been reported that fetal kidneys may have a lower immunogenicity than adult-type kidneys ^{30, 31}. Furthermore, transplanted fetal kidneys comprise recipient-derived vessels ^32–34, it is at least conceivable that rejection is reduced compared with organ transplantation using adult organs as donors. Notably, immunological advantage of this approach was that the recipient was a fetus, a specific characteristic of our technique. Transplantation during the fetal period, when the immune system is still developing, may have immunological advantages.

In xenogeneic transplantation into fetuses, the addition of a small amount of immunosuppressant resulted in longer-term engraftment (18 days). Previous studies have reported that cell transplantation can engraft in fetuses in utero in xenogeneic settings ^35–37. Although xenotransplantation faces a higher hurdle in terms of rejection compared with allogeneic transplantation, immunological advantages afforded by the abovementioned mechanisms were speculated.

One of the limitations of this study was that a kidney failure model was not used as the transplantation target. Because all recipient fetuses were normal (wild-type) fetuses, the actual therapeutic effect of fetal kidney transplantation could not be confirmed. In the future, we plan to perform transplantation verification using a congenital kidney disease model (Six2-expressing nephron progenitor cell depletion model) in rats. Furthermore, the CCr of a single MNB remained low, at 40–80 µL/min, compared with the CCr of 4,000 µL/min calculated from the 24-h urine collection of the neonates themselves at the same time point. To achieve therapeutic effects in the future, it is necessary to explore methods to enhance the function of a single MNB or to transplant multiple MNBs. For clinical application, technical investigations are also being carried out on pig fetuses as large animals. As the next stage in this research, immunological analyses will be performed using nonhuman primates as recipients.

The ultimate goal of this study was to develop a renal replacement therapy for children with severe congenital kidney diseases, particularly bilateral renal agenesis, for which there are few treatment options. In this study, we used MNBs as transplantation organs, established a transplantation method into fetuses, and demonstrated for the first time worldwide the success of transplantation into fetuses and the acquisition of functional organs capable of urine production in allogeneic settings. The superiority of fetal tissue as a donor organ has been previously pointed out; however, the present study suggests that the fetal elements on the recipient side may also have immunological and scaffold environment advantages. In this study, we successfully constructed an organ with urine production ability for as long as 150 days, which is considered a sufficient period as a bridge to dialysis in neonates, considering the lifespan of rodents. For application in humans, it is necessary to demonstrate this therapeutic effect using larger animal models, increase the number of transplanted MNBs, and improve the quality of the transplanted MNBs. This study is an important step toward the development of a new treatment approach for medical intervention in children with severe kidney diseases for which no effective treatment is available currently.

Research animals

The animal experiments performed here followed the Guidelines for the Proper Conduct of Animal Experiments of the Science Council of Japan (2006) and were approved by the Institutional Animal Care and Use Committee of the Jikei University School of Medicine (protocol numbers: 2023-021). All efforts were made to minimize animal suffering. Pregnant female SD rats, SD-Tg (CAG-enhanced green fluorescent protein [EGFP]) rats (GFP-SD rats), C57BL/6-Tg (CAG-EGFP) mice (GFP-B6 mice), and adult male SD rats were purchased from Sankyo Labo Service Corporation (Tokyo, Japan).

Isolation of fetal kidneys or MNBs

Pregnant rats and mice were anesthetized via the inhalation of isoflurane (2817774; Pfizer, New York, USA). The fetal kidneys or MNBs (E14.0–16.5 in rats and E13.0–13.5 in mice) were harvested, and the pregnant rats and mice were then killed immediately via an infusion of pentobarbital (120 mg/kg). All fetuses were killed by decapitation. Fetal kidneys or MNBs were dissected under a surgical microscope (M205FA; Leica Microsystems, Wetzlar, Germany).

Transplantation of rodent MNBs into the fetal subcutaneous space

A summary of this experiment is provided in Fig. 1a. GFP-SD rat MNBs (E14.0–16.5) (or GFP-B6 mouse fetal kidneys) were dissected and loaded onto the tip of a 15G or 16G needle (Saito Medical Instruments Inc., Tokyo, Japan) (Fig. 1b). The opposite end of the 15G or 16G needle was attached to a 0.5-mL syringe filled with HBSS (TERUMO, Tokyo, Japan). Pregnant SD rats (E18.0–18.5) were anesthetized via isoflurane inhalation. Subsequently, a midline incision was made, and the uterus was gently extracted from the abdominal cavity. The position of the fetuses was identified through the translucent uterine wall (Fig. 1c). Using the 15G or 16G needle loaded with GFP-SD rat MNBs (or GFP-B6 mice fetal kidneys), the uterine wall was punctured on the dorsal side of the fetus, and the needle was inserted directly into the fetal subcutaneous space under a stereomicroscope (Fig. 1d, e). The needle was advanced approximately 5 mm under the fetal skin, and 0.1–0.5 mL of HBSS was ejected to transplant the GFP-SD rat MNBs (or GFP-B6 mice fetal kidneys) into the fetal subcutaneous space. Immediately after transplantation, the needle was withdrawn. The presence of GFP-positive tissue was confirmed by fluorescence stereomicroscopy immediately after transplantation to ensure proper execution (Fig. 1f). To minimize the surgical duration, 2–4 fetuses per dam were used as recipients. The uterus was gently returned to the abdominal cavity. Approximately 5 mL of HBSS warmed to approximately 30°C was administered intraperitoneally to prevent adhesions. The anatomical positioning of the uterus within the abdominal cavity was confirmed, and the incision was closed.

Postnatal management

After natural birth at E22, the neonates were nursed by their biological mothers in the same cage. At postnatal day 0, the GFP-positive tissue was visualized on the body surface of the neonates using fluorescence microscopy. Neonates lacking the GFP-positive tissue were euthanized on the same day by decapitation. Body weight measurements were regularly conducted throughout the observation period. In addition, the GFP-positive tissue was visualized using fluorescence microscopy, and the longitudinal diameter was measured over time. When necessary, the transplanted fetal kidneys or MNBs were observed using transcutaneous ultrasonography (LOGIQ e Premium; GE HealthCare Technologies Inc, Chicago, USA). If a urinary cyst with a longitudinal diameter of 5 mm or more was detected via ultrasonography, it was deemed safe for percutaneous aspiration. Neonates were anesthetized by isoflurane inhalation, and a single puncture was made using a 23-29G needle (TERUMO, Tokyo, Japan) on the dorsal side to aspirate the urine. Single punctures were repeated at a frequency of 1–2 times per week, and the urine volume was measured.

Cryopreservation and thawing methods for fetal kidneys

The cryopreservation and thawing methods used for fetal kidneys have been reported previously (24 Matsui K. et al. Cryopreservation of fetal porcine kidneys for xenogeneic regenerative medicine. J Clin Med. 12, 2293 (2023)). Initially, the fetal kidneys were equilibrated in a base medium (MEMα supplemented with 20% fetal bovine serum [FBS; SH30070.03, HyClone Laboratories, Inc., Logan, UT, USA] and 1% antibiotic–antimycotic solution [15,240,062; Thermo Fisher Scientific, Waltham, MA, USA]) containing 7.5% ethylene glycol (EG; 055–00996; Wako, Osaka, Japan) and 7.5% dimethyl sulfoxide (DMSO; 317275-100ML; Millipore, Burlington, MA, USA) on ice for 15 min; followed by soaking in a base medium containing 15% EG and 15% DMSO on ice for an additional 15 min. Subsequently, the fetal kidneys were placed onto Cryotops (81111; Kitazato Corporation, Tokyo, Japan) and immediately submerged into liquid nitrogen for storage until transplantation. Fetal kidneys that had been cryopreserved for several weeks were thawed just prior to transplantation. The Cryotops containing fetal kidneys were swiftly transferred from liquid nitrogen to a base medium containing 1 M sucrose at 42°C for 1 min, then to a base medium containing 0.5 M sucrose at room temperature for 3 min, and finally washed twice in a base medium at room temperature for 5 min each time.

Transplantation of fetal kidneys or MNBs into adult rats

SD rats were anesthetized via isoflurane inhalation, and a laparotomy was performed through an abdominal midline incision. A pocket was created in the retroperitoneal space within the region bounded by the aorta, left ureter, and left renal artery using microtweezers (11253-25; Dumont, Montignez, Switzerland) under a surgical microscope. A single fetal kidney or MNB was transplanted into the pocket, which was subsequently closed using 10 − 0 nylon sutures (Muranaka Medical Instruments Co. Ltd., Osaka, Japan). The procedure was completed by closing the abdominal incision.

Biochemical measurements in the blood and urine

For blood tests, SD rats were anesthetized via isoflurane inhalation, and their tail veins were punctured using a 25G needle (TERUMO) to collect blood samples using hematocrit capillary tubes (2-454-21, AS ONE Corporation). The capillaries were sealed with wax (2-454-22, AS ONE Corporation) and centrifuged at 12,000 × g for 10 min to separate the serum from blood cells. The serum creatinine levels were quantified using a DRI-CHEM instrument (FUJIFILM). Urine samples were collected from MNBs and the rats’ 24-h urine collection. For the 24-h urine collection, SD rats were placed in rat metabolic cages (KN-650-MC, Sugiyama-gen Co., Ltd.) overnight. The urine samples were submitted to SRL, Inc. for the measurement of urine creatinine levels.

Histology and immunofluorescence

The harvested fetal kidneys or MNBs were fixed in 4% paraformaldehyde (161-20141, Wako) at 4°C overnight and subsequently embedded in paraffin. Sections with a thickness of 4 µm were obtained using a rotary microtome (HM355S, PHC, Tokyo, Japan). Standard procedures were used for staining with hematoxylin and eosin, Masson’s trichrome, and periodic acid–Schiff.

For immunostaining, the slides were deparaffinized, washed thrice in phosphate-buffered saline (PBS), and subjected to antigen retrieval by incubating with citrate buffer (#K 035; 10X Citrate Buffer, pH 6.0, DBS) at 121°C for 10 min. Following three additional washes in PBS, the slides were blocked at room temperature for 30 min using 5.0% skimmed milk. Subsequently, the sections were incubated overnight at 4°C with primary antibodies (Table 2), followed by incubation with secondary antibodies conjugated with Alexa Fluor 488, 546, or 647, as well as DAPI for 1 h, at room temperature. Finally, the sections were mounted with a glycerol-based liquid mounting medium. Each sample was examined under a fluorescence microscope (LSM880; Carl Zeiss, Oberkochen, Germany).

Table 2

List of primary antibodies.
Antigen	Host	Supplier	Cat. No.	Dilution
Nephrin	Guinea pig	Progen	GP-N2	1:100
LTL biotin	-	Vector	B-1325	1:200
E-cadherin	Mouse	Thermo	610181	1:100
E-cadherin	Rabbit	Cell Signaling	3195S	1:100
GFP	Rabbit	MBL	MBL598	1:500
GFP	Chicken	Abcam	ab13970	1:100
CD31	Goat	R&D	AF3628	1:100
CD3	Mouse	DAKO	Clone F7.2.38	1:100
CD3	Rabbit	Abcam	ab5690	1:100
Supplementary Table 1. Transplacental tacrolimus transfer.

Statistical analyses

All data are presented as the mean ± standard error of the mean. The data were analyzed using the two-tailed unpaired t-test and linear regression. A p-value of < 0.05 was considered statistically significant. Experimental data were analyzed using GraphPad Prism version 8.0 (GraphPad Software, Boston, MA, USA) and Microsoft Excel (Microsoft, Redmond, WA, USA). The details of the analyses are explained in each figure legend.

Competing interests

Author Eiji Kobayashi is the director of Kobayashi Regenerative Research Institute, LLC. Eiji Kobayashi has received a research fund from Sumitomo Pharma Co., Ltd., Osaka, Japan as a result of the Collaborative Research Agreement between The Jikei University School of Medicine and Sumitomo Pharma Co., Ltd. Author Eiji Kobayashi have received technical guidance fee from Fuji Micra Inc., Shizuoka, Japan. The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Additional information

Correspondence and requests for materials should be addressed to Shuichiro Yamanaka or Takashi Yokoo.

Author contributions

K. Morimoto wrote the main manuscript text. K. Morimoto and S. Yamanaka designed the study. K. Morimoto performed the experiments. K. Morimoto, S. Yamanaka, S. Yamamoto, N.K., Y.K., Y.I., K. Matsui, N.M., Y.S., T.T., T.F., S.F., S.T., K. Matsumoto, K.O., S.W., E.K., T.Y. interpreted the data and revised the manuscript. S. Yamanaka and T.Y. supervised the study. All authors reviewed the manuscript and granted permission to publish the study. T.Y. granted final permission to the manuscript.

Acknowledgments

We thank Ms. T. Tamatsukuri, Ms. T. Hayakawa, Ms. S. Kawagoe, and Ms. H. Gotoh for their experimental and technical assistance. This work was supported by the Japan Agency for Medical Research and Development (grant no. 22bm0704049h0003, 24bm1223003h0003 and 23bm1123036h0001], the Japan Society for the Promotion of Science (JSPS-KAKENHI; grant no. 21K08288 and 24K11439), and JST FOREST Program [grant no. JPMJFR2011].

Data availability statement

The main data supporting the results in this study are available within the paper and its Supplementary Information. All other data are available from the corresponding author upon reasonable requests.

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Yes there is potential Competing Interest. Author Eiji Kobayashi is the director of Kobayashi Regenerative Research Institute, LLC. Eiji Kobayashi has received a research fund from Sumitomo Pharma Co., Ltd., Osaka, Japan as a result of the Collaborative Research Agreement between The Jikei University School of Medicine and Sumitomo Pharma Co., Ltd. Author Eiji Kobayashi have received technical guidance fee from Fuji Micra Inc., Shizuoka, Japan. The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

SupplementaryFigure1.tif
Supplementary Fig. 1 a Structure of the fetal kidney before transplantation. b Periodic acid–Schiff staining revealed that the fetal kidney had not formed glomeruli or renal tubules before transplantation. Scale bars, 200 μm in (b). MNs, metanephroi
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Supplementary Video 1
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Fetal Kidney Transplantation into In Utero Fetuses

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Abstract

Figures

Introduction

Results

Maturation of transplanted GFP-SD rat MNBs in neonates

Development of the urine excretion method for GFP-SD rat MNBs transplanted into the subcutaneous space

Verification of the immunological advantages of fetal transplantation

Verification of xenotransplantation (mice to rats)

Discussion

Methods

Research animals

Isolation of fetal kidneys or MNBs

Transplantation of rodent MNBs into the fetal subcutaneous space

Postnatal management

Cryopreservation and thawing methods for fetal kidneys

Transplantation of fetal kidneys or MNBs into adult rats

Biochemical measurements in the blood and urine

Histology and immunofluorescence

Statistical analyses

Declarations

Competing interests

Additional information

Author contributions

Acknowledgments

Data availability statement

References

Additional Declarations

Supplementary Files

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