TONICITY MATURES THE COLLECTING DUCTS OF THE KIDNEY

The field of kidney organoids has flourished over the last decade. Methods have been developed to induce kidney organoids from mouse and human pluripotent stem cells. However, these kidney organoids are still immature compared to adult kidneys. In order to expand their applications, including disease modeling, drug development, and therapeutic transplantation, it is crucial to understand how the kidney matures during development. This is also a fundamental question in biology. Previous studies have reported that endocrine hormones, such as vasopressin and aldosterone, mature the collecting ducts (CDs) and that shear stress and metabolite supplementation promote the proximal tubule maturation. Our goal here was to discover a new maturation program. In particular, we hypothesized that high tonicity, a unique kidney characteristic, promotes CD maturation.

We isolated ureteric buds, the progenitors of CDs, from mouse embryos on embryonic day 11.5 and cultured them at high tonicity by adding sodium chloride to the culture medium. We then examined the maturity of these organoids in terms of their structure, gene expression, and function. To study the role of the transcription factor, NFAT5 (nuclear factor of activated T cells 5), we cultured ureteric buds isolated from CD-specific NFAT5 knockout mice. Based on these findings, we developed a maturation protocol for human CD organoids induced from induced pluripotent stem cells (iPSCs). To model congenital nephrogenic diabetes insipidus (NDI), we introduced a reported mutation into iPSCs, induced the mature CD organoids from them, and examined their responsiveness to vasopressin.

Mouse CDs altered their cellular morphology in response to high tonicity. Single-cell RNA sequencing (scRNA-seq) revealed that tonicity upregulated a broad spectrum of genes, including transcription factors, signaling molecules, and transporters. These genes accounted for approximately half of the maturation-associated genes of the medullary CDs. They became responsive to vasopressin at high tonicity. This maturation process was mainly dependent on NFAT5. In scRNA-seq analysis, mouse CD organoids cultured at high tonicity in the presence of vasopressin and aldosterone overlapped with postnatal day 7 medullary CDs with high expression levels of marker genes such as Aqp2 and Slc14a2. Additionally, we have established a maturation protocol for human CD organoids. These organoids also underwent morphological and transcriptional maturation and acquired vasopressin responsiveness at high tonicity. scRNA-seq analysis revealed that the mature CD organoids exhibited comparable expression levels of maturation markers to those of adult CDs. Finally, the mature CD organoids induced from iPSCs harboring a causative AVPR2 mutation of congenital NDI lost vasopressin responsiveness. In contrast, the healthy organoids responded to vasopressin with clear AQP2 expression in the apical membrane.

High tonicity is a driving force not only for urine concentration, but also for mammalian kidney maturation. Our mature CD organoids reached a postnatal stage, and potentially an adult stage. Our study uncovers a novel principle of kidney maturation triggered by environmental cues and offers a promising approach to maximize the potential of kidney organoids for future therapies.