Generation of patterned kidney organoids that recapitulate the adult kidney collecting duct system from expandable ureteric bud progenitors.

Current kidney organoids model development and diseases of the nephron but not the contiguous epithelial network of the kidney's collecting duct (CD) system. Here, we report the generation of an expandable, 3D branching ureteric bud (UB) organoid culture model that can be derived from primary UB progenitors from mouse and human fetal kidneys, or generated de novo from human pluripotent stem cells. In chemically-defined culture conditions, UB organoids generate CD organoids, with differentiated principal and intercalated cells adopting spatial assemblies reflective of the adult kidney's collecting system. Aggregating 3D-cultured nephron progenitor cells with UB organoids in vitro results in a reiterative process of branching morphogenesis and nephron induction, similar to kidney development. Applying an efficient gene editing strategy to remove RET activity, we demonstrate genetically modified UB organoids can model congenital anomalies of kidney and urinary tract. Taken together, these platforms will facilitate an enhanced understanding of development, regeneration and diseases of the mammalian collecting duct system.

3D kidney organoids for bench-to-bedside translation.

The kidneys are essential organs that filter the blood, removing urinary waste while maintaining fluid and electrolyte homeostasis. Current conventional research models such as static cell cultures and animal models are insufficient to grasp the complex human in vivo situation or lack translational value. To accelerate kidney research, novel research tools are required. Recent developments have allowed the directed differentiation of induced pluripotent stem cells to generate kidney organoids. Kidney organoids resemble the human kidney in vitro and can be applied in regenerative medicine and as developmental, toxicity, and disease models. Although current studies have shown great promise, challenges remain including the immaturity, limited reproducibility, and lack of perfusable vascular and collecting duct systems. This review gives an overview of our current understanding of nephrogenesis that enabled the generation of kidney organoids. Next, the potential applications of kidney organoids are discussed followed by future perspectives. This review proposes that advancement in kidney organoid research will be facilitated through our increasing knowledge on nephrogenesis and combining promising techniques such as organ-on-a-chip models.

Expansion of Human iPSC-Derived Ureteric Bud Organoids with Repeated Branching Potential.

Ureteric bud (UB) is the embryonic kidney progenitor tissue that gives rise to the collecting duct and lower urinary tract. UB-like structures generated from human pluripotent stem cells by previously reported methods show limited developmental ability and limited branching. Here we report a method to generate UB organoids that possess epithelial polarity and tubular lumen and repeat branching morphogenesis. We also succeed in monitoring UB tip cells by utilizing the ability of tip cells to uptake very-low-density lipoprotein, cryopreserving UB progenitor cells, and expanding UB tip cells that can reconstitute the organoids and differentiate into collecting duct progenitors. Moreover, we successfully reproduce some phenotypes of multicystic dysplastic kidney (MCDK) using the UB organoids. These methods will help elucidate the developmental mechanisms of UB branching and develop a selective differentiation method for collecting duct cells, contributing to the creation of disease models for congenital renal abnormalities.

Identification and characterization of cellular heterogeneity within the developing renal interstitium.

Kidney formation requires the coordinated growth of multiple cell types including the collecting ducts, nephrons, vasculature and interstitium. There is a long-held belief that interactions between progenitors of the collecting ducts and nephrons are primarily responsible for kidney development. However, over the last several years, it has become increasingly clear that multiple aspects of kidney development require signaling from the interstitium. How the interstitium orchestrates these various roles is poorly understood. Here, we show that during development the interstitium is a highly heterogeneous patterned population of cells that occupies distinct positions correlated to the adjacent parenchyma. Our analysis indicates that the heterogeneity is not a mere reflection of different stages in a linear developmental trajectory but instead represents several novel differentiated cell states. Further, we find that ß-catenin has a cell autonomous role in the development of a medullary subset of the interstitium and that this non-autonomously affects the development of the adjacent epithelia. These findings suggest the intriguing possibility that the different interstitial subtypes may create microenvironments that play unique roles in development of the adjacent epithelia and endothelia.

In developing mouse kidneys, orientation of loop of Henle growth is adaptive and guided by long-range cues from medullary collecting ducts.

The path taken by the loop of Henle, from renal cortex to medulla and back, is critical to the ability of the kidney to concentrate urine and recover water. Unlike most developing tubules, which navigate as blind-ended cylinders, the loop of Henle extends as a sharply bent loop, the apex of which leads the double tubes behind it in a 'V' shape. Here, we show that, in normal kidney development, loops of Henle extend towards the centroid of the kidney with an accuracy that increases the longer they extend. Using cultured kidney rudiments, and manipulations that rotate or remove portions of the organ, we show that loop orientation depends on long-range cues from the medulla rather than either the orientation of the parent nephron or local cues in the cortex. The loops appear to be attracted to the most mature branch point of the collecting duct system but, if this is removed, they will head towards the most mature collecting duct branch available to them. Our results demonstrate the adaptive nature of guidance of this unusual example of a growing epithelium, and set the stage for later work devoted to understanding the molecules and mechanisms that underlie it.

Morphology of the initial nephron-collecting duct connection in mice using computerized 3D tracing and electron microscopy.

Recently, the cellular origin of the connecting tubule (CNT) has been genetically characterized. The CNT is a segment between two embryonically different structures, the collecting duct originating from ureteric bud (UB), and the nephron derived from the cap mesenchyme. However, the cellular detail at the initial connection is limited. The present study demonstrated that the initial connection was composed of cells which were closely associated with the renal vesicle (RV), the initial nephron, and connected with the basal epithelium of the terminal UB tip at discrete points. The identification of the RV and UB tip was based on tracing of tubules on serial epoxy sections at mouse embryonic day 17.5. The cells at the initial connection were characterized by 1) irregularly-shaped nuclei and cells with cytoplasmic processes, 2) electron dense nuclei, 3) abundant intercellular spaces, 4) extensive cell-cell contacts with cell junctions, often zonulae adherences and occasionally focal fusion of opposing plasma membranes, and 5) numerous mitochondria, densely packed rosette-like polyribosomes, and widespread rER in the cytoplasm. Moreover, the tracing revealed that a terminal UB tip frequently connected to two nephrons at different developing stages. The UB tips, the initial connections, and the distal tubules of the S-shaped bodies did not express Na+-Cl- cotransporter, H+-ATPase, or aquaporin 2, while they were expressed in immature CNT of the capillary-loop stage nephrons throughout the kidney development. Consequently, the cells at the initial connection exhibit the morphological features suggestive of energy demanding, protein producing, and intercellular communicating. The cell morphology together with transporter development indicates that these cells serve several functions during the development of the initial connection, and that these functions are different from the cells' final functions as transportation.

Branching morphogenesis of the urinary collecting system in the human embryonic metanephros.

An elaborate system of ducts collects urine from all nephrons, and this structure is known as the urinary collecting system (UCS). This study focused on how the UCS is formed during human embryogenesis. Fifty human embryos between the Carnegie stage (CS) 14 and CS23 were selected from the Kyoto Collection at the Congenital Anomaly Research Center of Kyoto University, Japan. Metanephroses, including the UCS, were segmented on serial digital virtual histological sections. Three-dimensional images were computationally reconstructed for morphological and quantitative analyses. A CS timeline was plotted. It consisted of the 3-D structural morphogenesis of UCS and quantification of the total amount of end-branching, average and maximum numbers of generations, deviation in the metanephros, differentiation of the urothelial epithelium in the renal pelvis, and timing of the rapid expansion of the renal pelvis. The first UCS branching generation occurred by CS16. The average branching generation reached a maximum of 8.74 ± 1.60 and was already the twelfth in CS23. The total end-branching number squared between the start and the end of the embryonic period. UCS would reach the fifteenth branching generation soon after CS23. The number of nephrons per UCS end-branch was low (0.21 ± 0.14 at CS19, 1.34 ± 0.49 at CS23), indicating that the bifid branching occurred rapidly and that the formation of nephrons followed after. The renal pelvis expanded mainly in CS23, which was earlier than that reported in a previous study. The number of nephrons connected to the UCS in the expanded group (246.0 ± 13.2) was significantly larger than that of the pre-expanded group (130.8 ± 80.1) (P < 0.05). The urothelial epithelium differentiated from the zeroth to the third generations at CS23. Differentiation may have continued up until the tenth generation to allow for renal pelvis expansion. The branching speed was not uniform. There were significantly more branching generations in the polar- than in the interpolar regions (P < 0.05). Branching speed reflects the growth orientation required to form the metanephros. Further study will be necessary to understand the renal pelvis expansion mechanism in CS23. Our CS-based timeline enabled us to map UCS formation and predict functional renal capacity after differentiation and growth.

HNF1B controls epithelial organization and cell polarity during ureteric bud branching and collecting duct morphogenesis.

Kidney development depends crucially on proper ureteric bud branching giving rise to the entire collecting duct system. The transcription factor HNF1B is required for the early steps of ureteric bud branching, yet the molecular and cellular events regulated by HNF1B are poorly understood. We report that specific removal of Hnf1b from the ureteric bud leads to defective cell-cell contacts and apicobasal polarity during the early branching events. High-resolution ex vivo imaging combined with a membranous fluorescent reporter strategy show decreased mutant cell rearrangements during mitosis-associated cell dispersal and severe epithelial disorganization. Molecular analysis reveals downregulation of Gdnf-Ret pathway components and suggests that HNF1B acts both upstream and downstream of Ret signaling by directly regulating Gfra1 and Etv5 Subsequently, Hnf1b deletion leads to massively mispatterned ureteric tree network, defective collecting duct differentiation and disrupted tissue architecture, which leads to cystogenesis. Consistently, mRNA-seq analysis shows that the most impacted genes encode intrinsic cell-membrane components with transporter activity. Our study uncovers a fundamental and recurring role of HNF1B in epithelial organization during early ureteric bud branching and in further patterning and differentiation of the collecting duct system in mouse.

Talin regulates integrin ß1-dependent and -independent cell functions in ureteric bud development.

Kidney collecting system development requires integrin-dependent cell-extracellular matrix interactions. Integrins are heterodimeric transmembrane receptors consisting of α and ß subunits; crucial integrins in the kidney collecting system express the ß1 subunit. The ß1 cytoplasmic tail has two NPxY motifs that mediate functions by binding to cytoplasmic signaling and scaffolding molecules. Talins, scaffolding proteins that bind to the membrane proximal NPxY motif, are proposed to activate integrins and to link them to the actin cytoskeleton. We have defined the role of talin binding to the ß1 proximal NPxY motif in the developing kidney collecting system in mice that selectively express a Y-to-A mutation in this motif. The mice developed a hypoplastic dysplastic collecting system. Collecting duct cells expressing this mutation had moderate abnormalities in cell adhesion, migration, proliferation and growth factor-dependent signaling. In contrast, mice lacking talins in the developing ureteric bud developed kidney agenesis and collecting duct cells had severe cytoskeletal, adhesion and polarity defects. Thus, talins are essential for kidney collecting duct development through mechanisms that extend beyond those requiring binding to the ß1 integrin subunit NPxY motif.

Transcription factor TFCP2L1 patterns cells in the mouse kidney collecting ducts.

Although most nephron segments contain one type of epithelial cell, the collecting ducts consists of at least two: intercalated (IC) and principal (PC) cells, which regulate acid-base and salt-water homeostasis, respectively. In adult kidneys, these cells are organized in rosettes suggesting functional interactions. Genetic studies in mouse revealed that transcription factor Tfcp2l1 coordinates IC and PC development. Tfcp2l1 induces the expression of IC specific genes, including specific H+-ATPase subunits and Jag1. Jag1 in turn, initiates Notch signaling in PCs but inhibits Notch signaling in ICs. Tfcp2l1 inactivation deletes ICs, whereas Jag1 inactivation results in the forfeiture of discrete IC and PC identities. Thus, Tfcp2l1 is a critical regulator of IC-PC patterning, acting cell-autonomously in ICs, and non-cell-autonomously in PCs. As a result, Tfcp2l1 regulates the diversification of cell types which is the central characteristic of 'salt and pepper' epithelia and distinguishes the collecting duct from all other nephron segments.

Development and Diseases of the Collecting Duct System.

The collecting duct of the mammalian kidney is important for the regulation of extracellular volume, osmolarity, and pH. There are two major structurally and functionally distinct cell types: principal cells and intercalated cells. The former regulates Na+ and water homeostasis, while the latter participates in acid-base homeostasis. In vivo lineage tracing using Cre recombinase or its derivatives such as CreGFP and CreERT2 is a powerful new technique to identify stem/progenitor cells in their native environment and to decipher the origins of the tissue that they give rise to. Recent studies using this technique in mice have revealed multiple renal progenitor cell populations that differentiate into various nephron segments and collecting duct. In particular, emerging evidence suggests that like principal cells, most of intercalated cells originate from the progenitor cells expressing water channel Aquaporin 2. Mutations or malfunctions of the channels, pumps, and transporters expressed in the collecting duct system cause various human diseases. For example, gain-of-function mutations in ENaC cause Liddle's syndrome, while loss-of-function mutations in ENaC lead to Pseudohypoaldosteronism type 1. Mutations in either AE1 or V-ATPase B1 result in distal renal tubular acidosis. Patients with disrupted AQP2 or AVPR2 develop nephrogenic diabetes insipidus. A better understanding of the function and development of the collecting duct system may facilitate the discovery of new therapeutic strategies for treating kidney disease.

Elf5 is a principal cell lineage specific transcription factor in the kidney that contributes to Aqp2 and Avpr2 gene expression.

The mammalian kidney collecting ducts are critical for water, electrolyte and acid-base homeostasis and develop as a branched network of tubular structures composed of principal cells intermingled with intercalated cells. The intermingled nature of the different collecting duct cell types has made it challenging to identify unique and critical factors that mark and/or regulate the development of the different collecting duct cell lineages. Here we report that the canonical Notch signaling pathway components, RBPJ and Presinilin1 and 2, are involved in patterning the mouse collecting duct cell fates by maintaining a balance between principal cell and intercalated cell fates. The relatively reduced number of principal cells in Notch-signaling-deficient kidneys offered a unique genetic leverage to identify critical principal cell-enriched factors by transcriptional profiling. Elf5, which codes for an ETS transcription factor, is one such gene that is down-regulated in kidneys with Notch-signaling-deficient collecting ducts. Additionally, Elf5 is among the earliest genes up regulated by ectopic expression of activated Notch1 in the developing collecting ducts. In the kidney, Elf5 is first expressed early within developing collecting ducts and remains on in mature principal cells. Lineage tracing of Elf5-expressing cells revealed that they are committed to the principal cell lineage by as early as E16.5. Over-expression of ETS Class IIa transcription factors, including Elf5, Elf3 and Ehf, increase the transcriptional activity of the proximal promoters of Aqp2 and Avpr2 in cultured ureteric duct cell lines. Conditional inactivation of Elf5 in the developing collecting ducts results in a small but significant reduction in the expression levels of Aqp2 and Avpr2 genes. We have identified Elf5 as an early maker of the principal cell lineage that contributes to the expression of principal cell specific genes.

Prorenin receptor controls renal branching morphogenesis via Wnt/ß-catenin signaling.

The prorenin receptor (PRR) is a receptor for renin and prorenin, and an accessory subunit of the vacuolar proton pump H+-ATPase. Renal branching morphogenesis, defined as growth and branching of the ureteric bud (UB), is essential for mammalian kidney development. Previously, we demonstrated that conditional ablation of the PRR in the UB in PRRUB-/- mice causes severe defects in UB branching, resulting in marked kidney hypoplasia at birth. Here, we investigated the UB transcriptome using whole genome-based analysis of gene expression in UB cells, FACS-isolated from PRRUB-/-, and control kidneys at birth (P0) to determine the primary role of the PRR in terminal differentiation and growth of UB-derived collecting ducts. Three genes with expression in UB cells that previously shown to regulate UB branching morphogenesis, including Wnt9b, ß-catenin, and Fgfr2, were upregulated, whereas the expression of Wnt11, Bmp7, Etv4, and Gfrα1 was downregulated. We next demonstrated that infection of immortalized UB cells with shPRR in vitro or deletion of the UB PRR in double-transgenic PRRUB-/-/BatGal+ mice, a reporter strain for ß-catenin transcriptional activity, in vivo increases ß-catenin activity in the UB epithelia. In addition to UB morphogenetic genes, the functional groups of differentially expressed genes within the downregulated gene set included genes involved in molecular transport, metabolic disease, amino acid metabolism, and energy production. Together, these data demonstrate that UB PRR performs essential functions during UB branching and collecting duct morphogenesis via control of a hierarchy of genes that control UB branching and terminal differentiation of the collecting duct cells.

Integrin α3ß1 regulates kidney collecting duct development via TRAF6-dependent K63-linked polyubiquitination of Akt.

The collecting system of the kidney develops from the ureteric bud (UB), which undergoes branching morphogenesis, a process regulated by multiple factors, including integrin-extracellular matrix interactions. The laminin (LM)-binding integrin α3ß1 is crucial for this developmental program; however, the LM types and LM/integrin α3ß1-dependent signaling pathways are poorly defined. We show that α3 chain-containing LMs promote normal UB branching morphogenesis and that LM-332 is a better substrate than LM-511 for stimulating integrin α3ß1-dependent collecting duct cell functions. We demonstrate that integrin α3ß1-mediated cell adhesion to LM-332 modulates Akt activation in the developing collecting system and that Akt activation is PI3K independent but requires decreased PTEN activity and K63-linked polyubiquitination. We identified the ubiquitin-modifying enzyme TRAF6 as an interactor with the integrin ß1 subunit and regulator of integrin α3ß1-dependent Akt activation. Finally, we established that the developmental defects of TRAF6- and integrin α3-null mouse kidneys are similar. Thus K63-linked polyubiquitination plays a previously unrecognized role in integrin α3ß1-dependent cell signaling required for UB development and may represent a novel mechanism whereby integrins regulate signaling pathways.

The ureteric bud epithelium: morphogenesis and roles in metanephric kidney patterning.

The mammalian metanephric kidney is composed of two epithelial components, the collecting duct system and the nephron epithelium, that differentiate from two different tissues -the ureteric bud epithelium and the nephron progenitors, respectively-of intermediate mesoderm origin. The collecting duct system is generated through reiterative ureteric bud branching morphogenesis, whereas the nephron epithelium is formed in a process termed nephrogenesis, which is initiated with the mesenchymal-epithelial transition of the nephron progenitors. Ureteric bud branching morphogenesis is regulated by nephron progenitors, and in return, the ureteric bud epithelium regulates nephrogenesis. The metanephric kidney is physiologically divided along the corticomedullary axis into subcompartments that are enriched with specific segments of these two epithelial structures. Here, we provide an overview of the major molecular and cellular processes underlying the morphogenesis and patterning of the ureteric bud epithelium and its roles in the cortico-medullary patterning of the metanephric kidney.

Node retraction during patterning of the urinary collecting duct system.

This report presents a novel mechanism for remodelling a branched epithelial tree. The mouse renal collecting duct develops by growth and repeated branching of an initially unbranched ureteric bud: this mechanism initially produces an almost fractal form with young branches connected to the centre of the kidney via a sequence of nodes (branch points) distributed widely throughout the developing organ. The collecting ducts of a mature kidney have a different form: from the nephrons in the renal cortex, long, straight lengths of collecting duct run almost parallel to one another through the renal medulla, and open together to the renal pelvis. Here we present time-lapse studies of E11.5 kidneys growing in culture: after about 5 days, the collecting duct trees show evidence of 'node retraction', in which the node of a 'Y'-shaped branch moves downwards, shortening the stalk of the 'Y', lengthening its arms and narrowing their divergence angle so that the 'Y' becomes a 'V'. Computer simulation suggests that node retraction can transform a spread tree, like that of an early kidney, into one with long, almost-parallel medullary rays similar to those seen in a mature real kidney.

microRNA-dependent temporal gene expression in the ureteric bud epithelium during mammalian kidney development.

BACKGROUND: Our previous study on mouse mutants with the ureteric bud (UB) epithelium-specific Dicer deletion (Dicer UB mutants) demonstrated the significance of UB epithelium-derived miRNAs in UB development. RESULTS: Our whole-genome transcriptional profiling showed that the Dicer mutant UB epithelium abnormally retained transcriptional features of the early UB epithelium and failed to express many genes associated with collecting duct differentiation. Furthermore, we identified a temporal expression pattern of early UB genes during UB epithelium development in which gene expression was detected at early developmental stages and became undetectable by embryonic day 14.5. In contrast, expression of early UB genes persisted at later stages in the Dicer mutant UB epithelium and increased at early stages. Our bioinformatic analysis of the abnormally persistently expressed early genes in the Dicer mutant UB epithelium showed significant enrichment of the let-7 family miRNA targets. We further identified a temporal expression pattern of let-7 miRNAs in the UB epithelium that is anti-parallel to that of some early UB genes during kidney development. CONCLUSIONS: We propose a model in which the let-7 family miRNAs silence the expression of a subset of early genes in the UB epithelium at later developmental stages to promote collecting duct differentiation. Developmental Dynamics 244:444-456, 2015. © 2014 Wiley Periodicals, Inc.

Renal development: a complex process dependent on inductive interaction.

Renal development begins in-utero and continues throughout childhood. Almost one-third of all developmental anomalies include structural or functional abnormalities of the urinary tract. There are three main phases of in-utero renal development: Pronephros, Mesonephros and Metanephros. Within three weeks of gestation, paired pronephri appear. A series of tubules called nephrotomes fuse with the pronephric duct. The pronephros elongates and induces the nearby mesoderm, forming the mesonephric (Woffian) duct. The metanephros is the precursor of the mature kidney that originates from the ureteric bud and the metanephric mesoderm (blastema) by 5 weeks of gestation. The interaction between these two components is a reciprocal process, resulting in the formation of a mature kidney. The ureteric bud forms the major and minor calyces, and the collecting tubules while the metanephrogenic blastema develops into the renal tubules and glomeruli. In humans, all of the nephrons are formed by 32 to 36 weeks of gestation. Simultaneously, the lower urinary tract develops from the vesico urethral canal, ureteric bud and mesonephric duct. In utero, ureters deliver urine from the kidney to the bladder, thereby creating amniotic fluid. Transcription factors, extracellular matrix glycoproteins, signaling molecules and receptors are the key players in normal renal development. Many medications (e.g., aminoglycosides, cyclooxygenase inhibitors, substances that affect the renin-angiotensin aldosterone system) also impact renal development by altering the expression of growth factors, matrix regulators or receptors. Thus, tight regulation and coordinated processes are crucial for normal renal development.

[The cortical collecting duct plays a pivotal role in the kidney's local renin-angiotensin system]. / A gyujtocsatorna meghatározó szerepe a vese lokális renin-angiotenzin rendszerében.

The renin-angiotensin system is one of the most important hormone systems in the body, and the regulations as well as the role in the juxtaglomerular apparatus are well known. The present review focuses on renin secretion in a recently described localization, the cortical collecting duct. The authors display it in parallel of the copying strategy of an adult and a developing kidney. Furthermore, based on different animal studies it highlights the local role of renin released from the collecting duct. In chronic angiotensin II-infused, 2-kidney, 1-clip hypertensive model as well as in diabetic rats the major source of (pro)renin is indeed the collecting duct. In this localization this hormone can reach both the systemic circulation and the interstitial renin-angiotensin system components including the newly described (pro)renin receptor, by which (pro)renin is able to locally activate pro-fibrotic intracellular signal pathways. Consequently, one can postulate that in the future renin may serve either as a new therapeutic target in nephropathy associated with both hypertension and diabetes or as an early diagnostic marker in chronic diseases leading to nephropathy.

Prenatal programming of rat cortical collecting tubule sodium transport.

Prenatal insults have been shown to lead to elevated blood pressure in offspring when they are studied as adults. Prenatal administration of dexamethasone and dietary protein deprivation have demonstrated that there is an increase in transporter abundance for a number of nephron segments but not the subunits of the epithelial sodium channel (ENaC) in the cortical collecting duct. Recent studies have shown that aldosterone is elevated in offspring of protein-deprived mothers when studied as adults, but the physiological importance of the increase in serum aldosterone is unknown. As an indirect measure of ENaC activity, we compared the natriuretic response to benzamil in offspring of mothers who ate a low-protein diet (6%) with those who ate a normal diet (20%) for the last half of pregnancy. The natriuretic response to benzamil was greater in the 6% group (821.1 ± 161.0 µmol/24 h) compared with the 20% group (279.1 ± 137.0 µmol/24 h), consistent with greater ENaC activity in vivo (P < 0.05). In this study, we also directly studied cortical collecting tubule function from adult rats using in vitro microperfusion. There was no difference in basal or vasopressin-stimulated osmotic water permeability. However, while cortical collecting ducts of adult offspring whose mothers ate a 20% protein diet had no sodium transport (-1.9 ± 3.1 pmol·mm(-1)·min(-1)), the offspring of rats that ate a 6% protein diet during the last half of pregnancy had a net sodium flux of 10.7 ± 2.6 pmol·mm(-1)·min(-1) (P = 0.01) in tubules perfused in vitro. Sodium transport was measured using ion-selective electrodes, a novel technique allowing measurement of sodium in nanoliter quantities of fluid. Thus we directly demonstrate that there is prenatal programming of cortical collecting duct sodium transport.