Amniotic Fluid Stem Cells within Chimeric Kidney Rudiments Differentiate to Functional Podocytes after Transplantation into Mature Rat Kidneys

The pioneering work of Grobstein, Saxen, and Sariola led to the development of culture systems that could support the growth and differentiation of mouse kidney rudiments in vitro.¹ Cultured kidney rudiments can be easily manipulated, either genetically or by the application of growth factors, inhibitors, or other small biologic substances, and are thus useful model systems for investigating the mechanisms that regulate the development of the mammalian kidney. Kidney rudiments have also been evaluated for their potential to integrate into mature kidneys and treat renal insufficiency. Woolf et al.² showed that rudiments transplanted into neonatal mouse kidneys generated functional nephrons with filtering capacity, with a subsequent study by Rogers and Hammerman³ showing that transplanted rudiments could improve survival in anephric rats, albeit for only a few days. However, despite these promising results, another study by Ashton and co-workers⁴ showed that although the kidney rudiments continued to develop for a few weeks after transplantation, by 3 months, their level of maturity only reached that of early neonatal kidneys. This meant that the GFR of the transplanted rudiments was equivalent to only 2% of the GFR of an adult rat kidney, which explains why the rudiments were unable to sustain life in anephric rodents beyond a few days.

Although these studies suggest that kidney rudiments are unlikely to have much therapeutic value when transplanted into diseased kidneys, they can nevertheless be very effective tools for assessing the nephrogenic potential of various types of stem and progenitor cells. For instance, following the work of Atala and co-workers,⁵ who found that disaggregated metanephroi isolated from bovine embryos could self-organize to form functioning nephrons, Unbekandt and Davies⁶ developed an assay that involved disaggregating mouse kidney rudiments to single cells, combining them with exogenous stem/progenitor cells, and then allowing the cells to reaggregate to form chimeric kidney rudiments. These chimeric rudiments could be cultured in vitro, presenting excellent test systems for assessing whether the exogenous stem cells are capable generating specialized renal cells.⁶ Using this assay, it has been shown that certain types of stem cells, most notably pluripotent stem cells⁷ and amniotic fluid stem cells,⁸ are capable of integrating into developing renal structures and generating specialized renal cells, whereas other cell types, such as mesenchymal stem/stromal cells, are unable to do so,⁹ unless they have been engineered to overexpress glial cell line–derived neurotrophic factor (GDNF).¹⁰

The chimeric kidney rudiment assay, therefore, allows researchers to identify stem cell types with nephrogenic potential that could be used for applications in regenerative medicine, drug discovery, and disease modeling. However, although the chimeric rudiment assay is very useful for investigating whether different stem cell types can participate in nephron development and can be used with great effect to test the functionality of stem cell–derived proximal tubule cells,⁷ a major drawback with the in vitro kidney rudiment system is that endothelial cells do not invest the developing glomeruli, and consequently, there is an absence of capillary loops, resulting in the failure of podocyte maturation. Furthermore, the lack of vasculature means that the podocytes are unable to perform selective filtration as they would in vivo, and hence, the functionality of stem cell–derived podocytes cannot be tested. However, a study by Xinaris et al.¹¹ has shown that, if reaggregated rudiments, also referred to as kidney organoids, are transplanted into adult rat kidneys, they can generate vascularized glomeruli with capillary loops and slit diaphragms that seem to have some filtration capacity.

In this issue of the Journal of the American Society of Nephrology, Xinaris et al.¹² have extended these studies to (1) explore whether reaggregated mouse rudiments can perform ultrafiltration, (2) assess the nephrogenic potential of human amniotic fluid stem cells (hAFSCs) within chimeric rudiments comprised of reaggregated mouse metanephroi, and (3) determine whether the hAFSCs within the chimeras can generate mature, functional podocytes after transplantation into mature rat kidneys. Using electron microscopy, Xinaris et al.¹² first show that, after transplantation into the kidneys of uninephrectomized athymic adult rats, reaggregated mouse kidney rudiments could generate vascularized glomeruli, some of which contained mature podocytes with well developed foot processes and slit diaphragms. The functionality of the glomeruli was then investigated using fluorescently labeled dextrans of low (10 and 70 kD) and high (155 kD) molecular masses. Some of the nephrons that formed within the chimeric rudiments were found to be capable of performing ultrafiltration, which was evidenced by their ability to filter the low–molecular mass dextrans but exclude the high–molecular mass dextran. Xinaris et al.¹² then evaluated the nephrogenic potential of the hAFSCs. In contrast to a previous study,⁸ Xinaris et al.¹² showed that hAFSCs within chimeric rudiments cultured in vitro had a limited capacity to integrate into developing renal structures, but if genetically modified with an adenovirus vector encoding GDNF, they could become incorporated into the condensed metanephric mesenchyme. When chimeric rudiments containing GDNF-expressing hAFSCs were cultured for 1 day in vitro and then transplanted into mature rat kidneys, immunofluorescence analysis showed that the hAFSCs could readily form podocytes. The ability of the hAFSCs to form mature podocytes was confirmed using immunoelectron microscopy, which showed that the hAFSC-derived podocytes had well developed foot processes and slit diaphragms. Finally, to show functionality, dual immunogold staining for a human-specific antigen and BSA was performed to show that hAFSC-derived podocytes were able to endocytose albumin. An interesting finding of the study by Xinaris et al.¹² is that the hAFSCs had a marked propensity to form podocytes and rarely formed proximal tubule cells. The reasons for this are not entirely clear, but Xinaris et al.¹² obtained the same result using two different hAFSC lines, indicating that the tendency of the hAFSCs to form podocytes was not a peculiarity of any one particular hAFSC line.

An important aspect of the study by Xinaris et al.¹² is that it shows how transplanted chimeric rudiments could potentially be used to model diseases affecting human podocytes. For instance, the use of hAFSCs with mutations in genes that affect podocyte differentiation, growth, survival, and/or function would allow detailed studies of the processes of disease initiation and progression and the evaluation of possible therapies. Furthermore, by using patient–derived human induced pluripotent stem cells instead of hAFSCs, it would be possible to investigate a wider range of human kidney diseases because of the fact that, unlike hAFSCs, induced pluripotent stem cells cultured under the appropriate conditions can generate the full suite of cell types that comprise the nephron.¹³

Could chimeric kidney organoids be used to treat renal insufficiency? At present, this seems unlikely, because in addition to the aforementioned issues with low GFR that have been reported after the transplantation of intact rudiments,⁴ in the reaggregated chimeric rudiments, there is the additional problem of the ureteric bud tree not being continuous, making it unable to transfer urine from the corticomedullary region to the renal pelvis.

In conclusion, Xinaris et al.¹² report that if hAFSCs are engineered to express GDNF and then combined with disaggregated mouse kidney rudiments to form chimeric kidney organoids, the hAFSCs can differentiate to form functional podocytes after transplantation into adult rat kidneys. The technology used by Xinaris et al.¹² could potentially be used as a model for understanding diseases affecting the podocytes and a system for testing novel therapies to treat podocyte disease.

Disclosures

None.