Although progress has been made implanting early human kidney progenitors into the omentum of mice, the growth and function of these organoids have been significantly restricted 1 , 2 . In fact, these kidney progenitors have been shown to prolong the life of anephric rats for only 4 days beyond that of controls 3 . Moreover, these organoids have only vestigial renal arteries, veins and ureters, rendering the possibility of future clinical transplantation remote 1 , 2 . We hypothesize that while early embryonic kidneys can survive off of a small vascular supply from surrounding host tissue after implantation, a dedicated renal artery is needed to support future growth of the organ into a mature, life‐supporting kidney. However, to our knowledge no group has been able to transplant fetal organs into adult animal hosts due to the inherent mismatch between fetal and adult blood pressures. Here we introduce a device, an arterial flow regulator (AFR), to lower the perfusion pressure and rate of blood flow entering the transplanted human fetal organ from the adult host. Using the AFR, we transplanted human fetal kidneys aged 17–18 weeks in gestation into complement‐depleted Rag 2 KO rats. Human fetal kidney transplants exhibited remarkable growth in size and were capable of supporting the life of bilaterally nephrectomized rats.

Methods

Arterial flow regulator design and construction We custom built an inflatable silicone cuff that could be gas sterilized and reused after cleaning. A custom mold (Fabtec, San Luis Obispo, CA) was used to create a watertight silicone sleeve that could contain saline in its lumen. A 15 cm long silicone tube was integrated into the lumen of the sleeve while a luer lock device was attached to the distal end of the tube. The cuff was designed to fit around the human fetal renal artery and contained a discontinuity to allow the renal artery to slide into the cuff. The discontinuity was approximated together using a metal clip (McMaster Carr, Elmhurst, IL) that surrounded the entire cuff assembly. A syringe containing saline was attached to the luer lock to both inject and withdraw predetermined volumes of saline as needed.

Human fetal kidney procurement and preparation We obtained 55 fully intact human fetal kidneys gestational age 17–18 weeks (StemExpress, Placerville, CA). All human kidneys were deidentified, with patient consent and Institutional Review Board approval obtained by StemExpress. Every experiment involving human fetal tissues was in accordance with the Helsinki Declaration of 1975 and reviewed by Ganogen's Human Fetal Transplantation Research Ethics Committee consisting of two board‐certified transplant surgeons at separate academic centers, two members of the general public not affiliated with the institution and one board‐certified general clinician (Approval No. G001, G002 and G003).

Human fetal kidney transplantation All experiments involving animal subjects were performed in full compliance with the Animal Welfare Act and approved by Ganogen's Institutional Animal Use and Care Committee (Approval No. G0001). No federal, state or local government funds were used or applied toward any of the experiments involving animals or human fetal tissues. One day prior to surgery, Rag 2 KO rats (TGRS4410; Sigma Aldrich, St. Louis, MO) were given 500 µg/kg cobra venom factor (A600; Quidel, Santa Clara, CA) intraperitoneally. C3 complement depletion was confirmed using a radial immunodiffusion kit (RN023.3, The Binding Site, San Diego, CA). Rats were anesthetized with 1–3% isoflurane (1001936060; Baxter, Deerfield, IL) and placed within a sterile field under a surgical microscope. A midline abdominal incision was made and the abdominal aorta and inferior vena cava were carefully exposed and dissected from each other using fine tweezers. Vascular clamps (RS‐ 5420; Roboz, Gaithersburg, MD) were placed above and below the intended site of anastomosis on the aorta and vena cava. A patch of the fetal aorta connected to the renal artery and a patch of the fetal vena cava attached to the renal vein were anastomosed end‐to‐side to the rat's abdominal aorta and vena cava, respectively, using interrupted 11–0 nylon sutures (T4A00N07; AROSurgical, Newport Beach, CA). A patch of the human fetal bladder connected to the ureter was anastomosed to the rat's bladder using interrupted 8–0 nylon sutures (VT6A08N14; AROSurgical). Prior to the release of the vascular clamps, the AFR and a flow probe (0.7PSB; Transonic, Ithaca, NY) were placed on the human fetal renal artery. Upon resumption of blood flow through the arterial anastomosis, saline in the AFR was titrated to allow an average flow of around 2 mL/min. To measure blood pressure in the renal artery, a pressure transducer (FTH‐ 1211B‐0018; Transonic Systems) was placed in the renal artery past the AFR. Postoperatively, the animals were housed in sterile conditions and administered Rimadyl 5 mg/kg (Pfizer, New York City, NY) subcutaneously and trimethoprim‐sulfamethoxazole in their water. For BrdU pulse chase experiments, rats were injected subcutaneously on days 8–15, 23–30 and 38–45 posttransplantation with 100 mg/kg BrdU per day (B5002; Sigma‐Aldrich).

Kidney growth and nephron counting To assess growth and maturation of developing human fetal kidneys, three transplanted kidneys were removed every 15 days during a period of 45 days. Growth was evaluated by measuring weight and volume through water displacement according to standard procedures. Medullary ray glomerular counting was used to estimate nephron number as previously described 4.

Histology, immunohistochemistry and immunofluorescence For BrdU immunostaining, slides were deparaffinized and bathed in 2 N HCl (Sigma‐Aldrich 653799) for 1 h at 37°C followed by neutralization in 0.1 M Borate buffer and permeabilization with 0.3% Triton X‐100 (X100; Sigma‐Aldrich). The slides were then blocked with 10% goat serum (50–062Z; Life Technologies, Grand Island, NY) and incubated with rat monoclonal anti‐Brdu antibodies (ab6326; Abcam) at a 1:20 dilution overnight. Secondary anti‐rat antibodies conjugated to HRP (BA‐4000; Vector Labs, Burlingame, CA) were then added with DAB brown as the chromogen (SK‐4100; Vector Labs). For CD56 and CD24 immunostaining, slides were deparaffinized and bathed in 0.1% Triton X‐100 and blocked with 10% goat serum. Mouse monoclonal anti‐CD56 antibody (07–5603; Life Technologies) and rabbit polyclonal anti‐CD24 antibody (ab110; Abcam, Cambridge, UK) were then added and incubated with the slides overnight (one primary antibody per slide). Secondary anti‐rabbit antibodies conjugated to HRP (MP‐7401; Vector Labs) were added to slides containing anti‐CD24 antibodies while anti‐mouse antibodies conjugated to HRP (MP‐7500; Vector Labs) were added to slides containing anti‐CD56 antibodies. Finally, DAB brown chromogen (SK‐4100; Vector Labs) was added to the slides. For CD31 immunostaining, slides were deparaffinized and bathed in 0.1% Triton X‐100 and blocked with 10% goat serum. Mouse monoclonal human‐specific anti‐CD31 antibody (M0823; Dako, Carpenteria, CA) and anti‐ pan rat endothelium (ab9774; Abcam) were then added and incubated with the slides overnight. Secondary anti‐mouse antibodies conjugated to HRP (MP‐ 7500, Vector Labs) and secondary anti‐rat antibodies conjugated to HRP (MP‐ 7444–15; Vector Labs) were added to the slide sequentially. For immunofluorescence Na+/K+‐ATPase and CD31 co‐staining, frozen slides were washed in PBS and bathed in 0.1% Triton X‐100. Primary mouse monoclonal anti‐ Na+/K+‐ATPase antibodies (Abcam) and rabbit polyclonal anti‐CD31 antibodies were incubated with the slides overnight at 4°C. Secondary goat anti‐mouse antibodies conjugated to Alexa Fluor 488 and donkey anti‐rabbit antibodies conjugated to Alexa Fluor 594 (Life Technologies) were added to the slides. A DAPI counterstain was then added and the slides were examined under a fluorescent microscope (Leica, Wetzlar, Germany).