Formation of Mature Nephrons by Implantation of Human Pluripotent Stem Cell-Derived Progenitors into Mice.

In the future, stem cell-based technologies may be harnessed to replace conventional dialysis and transplantation in patients with diabetic nephropathy. Recently, there has been considerable effort to improve methods for the differentiation of human pluripotent stem cells (hPSCs) into kidney cells in culture. Here, we present a protocol for obtaining more advanced kidney structures than have currently been possible in vitro, including vascularized glomeruli and tubular elements. HPSCs are first differentiated in 2D culture to a kidney progenitor stage. These cells are then dissociated and injected subcutaneously into immunocompromised mice. Twelve weeks later, the cells have developed into mature kidney structures and are excised for further characterization. This method constitutes a significant improvement on protocols that involve either exclusively a 2D culture or placing the cells in 3D organoid culture at the air-liquid interface in vitro.

Estimated Nephron Number of the Donor Kidney: Impact on Allograft Kidney Outcomes.

BACKGROUND: Low birth weights have been associated with a reduction in nephron number with compensatory hypertrophy of existing glomeruli. The impact of donor birth weight as an estimate of nephron number on allograft function, however, has not been examined. METHODS: We collected donor birth weight, kidney weight, and volume from 91 living kidney donor-recipient pairs before nephrectomy and after 12, 36, and 60 months. Nephron number was calculated from donor birth weight and age. RESULTS: Donor birth weight, kidney weight/body surface area (BSA), and kidney volume showed a moderate positive correlation with allograft estimated glomerular filtration rate (eGFR) at 12 months (P < .05). Donor age showed a negative moderate correlation with allograft eGFR at 12 months (P = .015). The strongest correlation with allograft eGFR was observed for calculated donor kidney nephron number at 12, 36, and 60 months (R, 0.340, 0.305, and 0.476, respectively; P < .05). No impact was observed on allograft daily proteinuria of any investigated marker (P > .05). Recipients of donors with birth weight <2.5 kg had need of a significantly greater number of antihypertensive drugs (P < .05). CONCLUSIONS: Calculated nephron number from donor birth weight and age is suggested to be superior to donor kidney weight/BSA and volume regarding allograft function. Calculated nephron number could estimate expected eGFR and guide decision making in cases of impaired allograft function.

Validity of surrogate measures for functional nephron mass.

BACKGROUND: Transplanted nephron mass is an important determinant of long-term allograft survival, but accurate assessment before organ retrieval is challenging. Newer radiologic imaging techniques allow for better determination of total kidney and cortical volumes. METHODS: Using volume measurements reconstructed from magnetic resonance or computed tomography imaging from living donor candidates, we characterized total kidney (n=312) and cortical volumes (n=236) according to sex, age, weight, height, body mass index (BMI), and body surface area (BSA). RESULTS: The mean cortical volume was 204 mL (range 105-355 mL) with no significant differences between left and right cortical volumes. The degree to which existing anthropomorphic surrogates predict nephron mass was quantified, and a diligent attempt was made to derive a better surrogate model for nephron mass. Cortical volumes were strongly associated with sex and BSA, but not with weight, height, or BMI. Four prediction models for cortical volume constructed using combinations of age, sex, race, weight, and height were compared with models including either BSA or BMI. CONCLUSIONS: Among existing surrogate measures, BSA was superior to BMI in predicting renal cortical volume. We were able to construct a statistically superior proxy for cortical volume, but whether relevant improvements in predictive accuracy could be gained needs further evaluation in a larger population.

Cellular transplantation of nephrons.

Cellular transplantation of nephrons. Embryonic renal cellular primordia transplanted into animal hosts undergo nephrogenesis in situ, become vascularized by blood vessels of host origin, exhibit excretory function, and support life in otherwise anephric hosts. Renal primordia can be transplanted across isogeneic, allogeneic, and both concordant (rat to mouse) and highly disparate (pig to rodent) xenogeneic barriers. Here I review studies exploring the therapeutic potential for renal organogenesis posttransplantation of cellular kidney primordia.

Engraftment of human early kidney precursors.

Kidney transplantation has been one of the major medical advances of the past 30 years; however, it is becoming increasingly apparent that the supply of organs is limited and will not improve with current medical practice. This review summarizes recent data whereby precursors of the adult kidney found in embryos and fetal tissue have been grafted into murine hosts to examine their feasibility as an alternative source for renal transplantation. When obtained at specific time points during human gestation, kidney precursors meet with specific demands; they grow tremendously, differentiate exclusively along the nephric lineage with no evidence of malignant transformation, become vascularised, to a larger extent, by host vessels, and produce urine in host animals. In addition, they exhibit decreased immunogenicity compared to adult counterparts. Organogenesis is best achieved when utilizing early undifferentiated progenitors rather than later-gestation kidneys. Nevertheless, in order for these transplants to be applicable for human transplantation, both a functional urinary anastomosis and derivation of blood supply sufficient to correct biochemical abnormalities remain to be established.

A strategy for in vitro propagation of rat nephrons.

BACKGROUND: Recent advances in the understanding of the molecular biology of rodent renal development have lead to the ability to culture the components of the developing rat kidney-the ureteric bud (UB) and the metanephric mesenchyme (MM)-in isolation from one another. Here we here describe a method for subculturing and propagating either whole rat metanephric rudiments or isolated rat UBs. Exploiting the branching program intrinsic to the UB, propagated rat UBs can be recombined with fresh rat mesenchyme to form a large number of rat "neokidneys" derived from a single progenitor that may be amenable to site-specific modulation of function. METHODS: Whole rat metanephric rudiments or isolated rat UBs were cultured and subdivided through several generations. Both cultured progenitor and subsequent generations of isolated rat UBs were recombined with freshly isolated rat metanephric mesenchyme. The tubules of these rat neokidneys were examined for expression of epithelial markers. RESULTS: Isolated rat UBs and whole rat metanephric rudiments could be propagated through several generations and appeared morphologically identical to their progenitors. Generations of isolated rat UB could be recombined with fresh rat mesenchyme and the resultant neokidney displayed the same morphologic appearance as the whole rat kidney rudiment. The UB-derived and MM-derived portions of the tubules of these rat neokidneys appear contiguous. CONCLUSIONS: The recombination of cultured and propagated rat UB with rat mesenchyme yielded rat neokidneys with tubular structures that appeared morphologically identical to whole rat kidney. In vitro propagation of rat metanephric rudiments and recombination of rat UB and MM suggest the possibility of designing nephrons that possess specific desirable functions that can be propagated in vitro.

Glomerular laminin isoform transitions: errors in metanephric culture are corrected by grafting.

Glomerular basement membrane (GBM) assembly and maturation are marked by the replacement of laminin-1 (containing alpha 1-, beta 1-, and gamma 1-chains) with laminin-11 (consisting of alpha 5-, beta 2-, and gamma 1-chains). Similarly, the alpha 1- and alpha 2-chains of type IV collagen are replaced by collagen alpha 3-, alpha 4-, and alpha 5(IV)-chains. The cellular origins of these molecules and mechanisms for isoform removal and substitution are unknown. To explore glomerular laminin isoform transitions in vitro, we assessed metanephric organ cultures. Standard culture conditions do not support endothelial cell differentiation, and glomerular structures that form in vitro are avascular. Nevertheless, extensive podocyte development occurs in these cultures, including the formation of foot processes and assembly of a GBM-like matrix. Here, we show that the podocyte-specific markers, glomerular epithelial protein 1 and nephrin, which are normally expressed in capillary loop stage glomeruli in vivo, are also expressed by glomerular figures that form in organ culture. However, the GBM-like segments that form in vitro do not undergo normal laminin isoform switching. Instead, both laminin alpha 1- and alpha 5-chains are present, as is the beta 1-chain, but not beta 2. When avascular organ-cultured kidneys are grafted into anterior eye chambers, however, kidney-derived angioblasts establish extensive vasculature by 6 days, and glomeruli are lined by endothelial cells. We evaluated embryonic day 12 (E12) vascular endothelial growth factor receptor (Flk1)-lacZ kidneys that had first been grown in organ culture for 6--7 days and then grafted into wild-type mice. Correct laminin isoform substitution occurred and correlated with the appearance of endothelial cells expressing Flk1. Our findings indicate that endothelial cells, and/or factors present in the circulation, mediate normal GBM laminin isoform transitions in vivo.

Distinguished Scientists Lecture Series. New developments in kidney development.

BACKGROUND/AIMS: The number of kidney transplantations performed per year is limited due to the availability of donor organs. One possible solution to the organ shortage is the use of renal xenografts. However, the transplantation of xenografts is complicated by rejection. METHODS: It has been postulated that the host immune response might be attenuated following the transplantation of embryonic kidneys (metanephroi) rather than developed (adult kidneys). Transplanted metanephroi become chimeric organs in that their blood supply originates from the host. It is possible to transplant a developing metanephros, without the use of immunosuppression, from one outbred rat to another. RESULTS: Transplanted metanephroi grow, develop, become vascularized, and function in host rats. In contrast, developed adult kidneys transplanted from one rat to another undergo rejection within 7 days after transplantation. CONCLUSIONS: These observations suggest that metanephric tissue may be less immunogenic than adult kidney. Transplantation of metanephroi represents a new development that could lead to a novel therapeutic approach to the treatment of chronic renal failure.

Transplantation of developing metanephroi into adult rats.

BACKGROUND: Transplantation of developing metanephroi into adult hosts has been proposed as a means to augment host renal function. METHODS: We implanted whole metanephroi from embryonic day 15 (E15) rats subcapsularly in kidneys or into the omentum of non-immunosupressed adult rat hosts. At the time of implantation, some host rats underwent unilateral nephrectomy (UNX) or unilateral nephrectomy and partial contralateral renal infarction (1 1/2 NX). E15 metanephroi contained only metanephric blastema, segments of ureteric bud, and primitive nephrons with no glomeruli. RESULTS: Four to six weeks post-implantation, metanephroi from E15 rats had enlarged, become vascularized, and had formed mature tubules and glomeruli. Ureters of metanephroi transplanted into the omentum were anastomosed to hosts' ureters that remained after UNX. Four weeks following ureteroureterostomy, the contralateral kidney was removed. Inulin clearances of seven metanephroi implanted into UNX hosts averaged 0.11 +/- 0.02 microliters/min/100 g (2.42 +/- 0.70 microliters/min/g kidney wt) and the creatinine clearances averaged 0.65 +/- 0.18 microliters/min/100 g. Metanephroi weighed 71 +/- 15 mg (approximately 4% of the contralateral native kidney). The transplanted metanephroi were vascularized by arteries originating from the omentum. Both weights of transplanted metanephroi (145 +/- 24 mg) and inulin clearances of transplanted metanephroi (30.1 +/- 8.7 microliters/min/g kidney weight) were significantly increased in rats that underwent 1 1/2 NX compared to UNX. In contrast, transplantation of developed kidneys resulted in rejection. CONCLUSIONS: Our findings establish that functional chimeric kidneys develop from metanephroi transplanted in adult hosts.

Integration of new embryonic nephrons into the kidney.

The current report summarizes our experiments exploring the feasibility of creating a chimeric kidney, that is, an organ constituted by cells derived from more than one fertilized ovum. The overall strategy has been to obtain donor renal tissue from avian and murine embryos and to implant this into the host avian mesonephric mesoderm or into the cortex of murine neonatal kidney. In both models, donor cells were distinguished from the host by the presence of characteristic nuclear or cytoplasmic markers. Examination of quail to chick transplants showed the tandem development of mesonephric tissue in the form of bilobed organ. In the mouse chimeric kidney, examined 2 to 4 weeks postnatally, transplanted metanephric tissue grew and developed glomeruli, proximal tubules, and cords of cells, which extended into the medulla of the host kidney. Before death, intravenous FITC-dextran was administered to the host mouse. Some transplanted tubules were connected to filtering glomeruli, as judged by the presence of fluorescein within their lumens. These experimental models provide novel means with which to study nephrogenesis in vivo. Finally, if the embryonic donor tissue could be genetically engineered before implantation, the prospect of "nephron therapy" arises, in which altered implanted nephrons could deliver therapeutically useful molecules into the urine or kidney interstitium.

Creation of a functioning chimeric mammalian kidney.

The possibility of adding new nephrons to the mammalian kidney was studied. Embryonic metanephric tissue was implanted into the renal cortex of neonatal mice less than 24 hours old, and the development of the chimeric kidney was followed over the following two to four weeks. Donor tissue was obtained from the homozygous beige mouse and a mouse line transgenic for the beta-globin gene, which provided distinct cellular and nuclear markers which were used to distinguish donor from recipient nephrons. Differentiation and growth of donor nephrons occurred in the host kidney and included vascularized glomeruli, mature proximal tubules, and tubular extensions into the renal medulla. Glomerular filtration was demonstrable in donor nephrons using FITC-dextran as a marker of filtration into the proximal tubules. Transplantation of metanephric tissue into adult mouse kidneys did not lead to glomerular or tubular differentiation. This study demonstrates the feasibility of adding functioning nephrons to mammalian kidneys in species in which there is ongoing nephrogenesis post-natally.