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American Journal of Physiology - Renal Physiology logoLink to American Journal of Physiology - Renal Physiology
. 2023 Sep 28;325(6):F695–F706. doi: 10.1152/ajprenal.00166.2023

Navigating the kidney organoid: insights into assessment and enhancement of nephron function

Nahid Tabibzadeh 1,2, Lisa M Satlin 3, Sanjay Jain 4,5,6, Ryuji Morizane 1,2,
PMCID: PMC10878724  PMID: 37767571

graphic file with name f-00166-2023r01.jpg

Keywords: development, function, kidney, organoid, physiology

Abstract

Kidney organoids are three-dimensional structures generated from pluripotent stem cells (PSCs) that are capable of recapitulating the major structures of mammalian kidneys. As this technology is expected to be a promising tool for studying renal biology, drug discovery, and regenerative medicine, the functional capacity of kidney organoids has emerged as a critical question in the field. Kidney organoids produced using several protocols harbor key structures of native kidneys. Here, we review the current state, recent advances, and future challenges in the functional characterization of kidney organoids, strategies to accelerate and enhance kidney organoid functions, and access to PSC resources to advance organoid research. The strategies to construct physiologically relevant kidney organoids include the use of organ-on-a-chip technologies that integrate fluid circulation and improve organoid maturation. These approaches result in increased expression of the major tubular transporters and elements of mechanosensory signaling pathways suggestive of improved functionality. Nevertheless, continuous efforts remain crucial to create kidney tissue that more faithfully replicates physiological conditions for future applications in kidney regeneration medicine and their ethical use in patient care.

NEW & NOTEWORTHY Kidney organoids are three-dimensional structures derived from stem cells, mimicking the major components of mammalian kidneys. Although they show great promise, their functional capacity has become a critical question. This review explores the advancements and challenges in evaluating and enhancing kidney organoid function, including the use of organ-on-chip technologies, multiomics data, and in vivo transplantation. Integrating these approaches to further enhance their physiological relevance will continue to advance disease modeling and regenerative medicine applications.

INTRODUCTION

The kidneys play a critical role in maintaining the internal environment of organisms by maintaining fluid and electrolyte balance, and eliminating toxins (1). The nephron of the kidney, composed of the glomerulus and the renal tubules, is surrounded by a stroma containing fibroblasts and immune cells (2, 3) and is intimately associated with a highly organized vasculature (4). These cells and structures are uniquely organized in different regions along the cortico-medullary axis of the kidney (5, 6). Within the nephrons, the glomeruli produce ∼180 L of ultrafiltrate per day (5), which then undergoes reabsorption and secretion processes in sequential tubular segments to produce the final urine that is expelled into the renal pelvis (6). This mature organization of nephrons in each adult kidney with highly specialized anatomical organization requires, early in organogenesis, reciprocal inductive interactions between progenitor cells of the ureteric bud and metanephric mesenchyme (7). Developing model systems that recapitulate the functions of the human kidney holds great promise for understanding the mechanisms of functional decline in the kidney, as well as strategies to restore healthy function in disease states and replace damaged kidneys (8). This area of research is of significant interest to the kidney community, including patients and nephrologists.

As our understanding of kidney organoids continues to expand, one of the key questions that arise is regarding their functional capacity when generated in vitro. Researchers in the field of organoid development aim to cultivate three-dimensional (3-D) structures from pluripotent stem cells that can differentiate and mimic the structure and function of a mammalian kidney. Current kidney organoids partially replicate the complexity of renal tissue by containing important components such as endothelial cells, stromal cells, podocytes, and tubules. The proportions of each cell type vary depending on the specific differentiation protocol used (9). However, their ability to function and form a connected system for urine drainage remains largely unknown. Claude Bernard, a renowned physiologist, famously stated, “The function of an organ or apparatus should be determined by its structure” (10). Yet, this first needs to be confirmed with proper functional evaluation in a new technology such as kidney organoids. In alignment with the RBK Consortium’s goal “to coordinate and support studies that will result in the ability to generate or repair nephrons that can function within the kidney” (11), a Functional Assay Working Group was established to catalog functional assays optimized to demonstrate successful function in organoids and other renal model systems, as mentioned in previous reviews (12, 13). In this perspective, we provide an overview of the recent advances in the characterization and optimization of the functional capacity of organoids, including strategies that replicate physiological processes by stimulating their maturation. Based on the results of these studies, the path forward must be prioritized to identify mechanisms to accelerate the functional maturation of organoids in the future.

STRUCTURE AND FUNCTION OF THE KIDNEYS AND THE KIDNEY ORGANOIDS: HOW DO THEY COMPARE?

Several experimental procedures have been established to generate kidney organoids, resulting in differences in structure and cellular composition (9, 1421). However, they share common phenotypic features including cellular types and structures found in mammalian kidneys (Fig. 1A). To confirm the physiological relevance of kidney organoids, characterization of its structural organization and the function of each specific segment is necessary. An inherent limitation to the functional analysis pertains to the 3-D configuration of these miniature organs lacking a proper inflow and outflow. These are theoretically necessary for the study of filtration, reabsorption, and secretion processes. Research groups have aimed to overcome these challenges by using various assays, and the key functional findings related to each segment are summarized in Fig. 2 and in this section.

Figure 1.

Figure 1.

Kidney organoid structures and cellular components. A: three-dimensional (3-D) images of kidney organoids on day 49 of differentiation. B and C: immunostaining displaying organoid glomeruli (B) and tubules (C). D: an electron microscope image showing brush border-like structures (#) and mitochondria (*) in organoid tubules. E: immunofluorescence microscopy displaying segments of proximal, loops of Henle, and distal tubules. F: single-nuclear RNA sequencing of healthy human kidneys from the Human Biomolecular Atlas Program (HuBMAP) and Kidney Precision Medicine Project (KPMP) revealing the expression of cadherin-1 (CDH1) in proliferating proximal tubules, loops of Henle, and distal nephrons. G: immunostaining showing organoid vasculature and stromal cells. The images of A were kindly provided by Ken Hiratsuka, BE were from Morizane et al. (14), and G were from Gupta et al. (22).

Figure 2.

Figure 2.

Functions identified in the kidney organoids in each nephron segment. An illustration summarizing studies of kidney organoid function in each nephron segment is shown. BKCa, large-conductance Ca2+-activated K+ channel; EB, ethidium bromide; ENaC, epithelial Na+ channel; FPE, fluid-phase endocytosis; OCT2, organic cation transporter 2; RBE, receptor-based endocytosis; Rho123, rhodamine 123; ROMK, renal outer medullary K+ channel; 6CF, 6-carboxyfluorescein.

Glomerulus

Structure in kidney organoids.

Kidney organoids display round-shaped structures with clustered cells expressing podocalyxin and nephrin indicative of podocytes, surrounded by a lumen separating the cluster from an epithelium resembling the parietal layer of the Bowman’s capsule. Foot processes have also been observed by electron microscopy (14). These glomerulus-like structures, however, usually lack polarity and vascularization that mimics the typical glomerular capillary tuft when cultured under static conditions. Along the same line, stromal cells suggestive of mesangial cells are usually not observed in these structures (Fig. 1B).

Glomerular filtration.

The glomerular filtration barrier within the fully differentiated mammalian kidney is composed of three layers arranged in multiple loops that confer a large area able to support the filtration of massive volumes of plasma. A specific fenestrated endothelium within the capillary loops is surrounded by the glomerular basement membrane, itself covered by the podocyte foot processes interconnected by slit diaphragms (8). Both cell types are arranged in close communication with the supporting mesangial cells and matrix. Due to Starling forces and the particular architecture of this barrier, plasma is filtered to produce ultrafiltrate containing water and solutes of low molecular weight. In kidney organoids maintained under static conditions, as this architecture is usually not seen, an effective filtration process is quite unlikely. To date, there is no reported evidence of glomerular filtration occurring within kidney organoids generated in vitro, except in cases where they have been transplanted into animals (summarized below in Response to In Vivo Transplantation) (21, 2328).

Proximal Tubule

Structure in kidney organoids.

A tubular structure with columnar epithelium is usually observed contiguous with these podocyte clusters. These tubular structures bind Lotus tetragonolobus lectin (LTL) along the brush border of their apical membranes, similar to observations made in the proximal tubule (PT) of adult human kidneys (18). Accordingly, ultrastructural analysis of this epithelium confirmed the presence of an apical brush border as well as cells densely packed with mitochondria (14), suggestive of the high energetic demand of PT cells (Fig. 1, C and D). Detailed ultrastructural analyses have not been performed to date to identify features indicative of discrete segments of the PT, namely S1 and S2 within the convoluted PT (pars convoluta) and S3, within the straight PT (pars recta) (29). It is thus not known if the current kidney organoid protocols achieve PT subsegment differentiation.

Proximal tubule absorption and secretion.

Due to its large surface area of exchange, the fully differentiated PT of the mammalian kidney is the site of bulk reabsorption of low-molecular-weight molecules, amino acids, glucose, water, phosphate, potassium, bicarbonate, and sodium (30). The segment notably accounts for the reabsorption of ∼70% of the filtered load of sodium and water both through the paracellular route, mediated by claudin-2, and through transcellular pathways, via multiple cotransporters and exchangers localized to the apical or basolateral membranes. On the other hand, low-molecular-weight molecules are reabsorbed through receptor-based endocytosis that is mediated by megalin and/or cubilin, or fluid-phase endocytosis. These two endocytic pathways of the PT are usually assessed by labeled cargo uptake, and in particular fluorescent low-molecular-weight dextran for fluid-phase endocytosis (31). The other central role of the PT is its secretory clearance of small molecules that are directly delivered by the peritubular capillaries to the basolateral side of the epithelium. Waste products and toxins, in particular protein-bound drugs that are not directly filtrated by the glomerulus, are mainly secreted via organic anion or cation transporters (OAT and OCT) (32). The transporters implicated in the reabsorption or the secretion of solutes are differentially expressed in the three segments of the PT. OCT2, for instance, exhibits higher expression levels in the S3 segment compared with the S1/S2 segments. It serves as the primary transporter of cisplatin at the basolateral membrane, which explains the drug’s accumulation in the PT. This accumulation is specifically responsible for the susceptibility of proximal tubules to cisplatin-induced nephrotoxicity (33).

Unlike the complex architecture of glomeruli, which has not been perfectly replicated under static conditions in kidney organoids, the LTL-positive tubular segments in organoids exhibit striking structural and ultrastructural similarities with PT. These similarities were confirmed by single-cell RNA sequencing revealing the expression of specific PT transporters and receptors within LTL-positive epithelium, such as cubilin and OAT1 (34). Several pieces of evidence strengthen the hypothesis of PT functionality in kidney organoids. Cargo uptake has been demonstrated specifically in this segment in several reports (15, 16, 21). Regarding PT drug uptake and secretion, Przepiorski et al. (35) reported in vitro OAT-dependent transport of six carboxyfluorescein (6-CF). Consistent with these findings, Rizki-Safitri et al. (36) demonstrated the transepithelial dynamic transit of rhodamine 123 by live imaging, from its basolateral OCT2-dependent uptake to its luminal multidrug resistance mutation 1 (MDR1)-dependent excretion. Through a protocol aiming at simultaneously increasing the nephron progenitor population and delaying nephron initiation, Vanslambrouck et al. (37) generated kidney organoids with an enhanced PT population able to increase the uptake of albumin. In addition, single-cell RNA sequencing data suggested the presence of different PT populations bearing some resemblance to S1, S2, and S3 segments. PT enhancement occurred, however, at the cost of a reduced distal tubule population compared with their standard differentiation protocol. Future efforts should be directed at more precisely evaluating these different processes along the length of LTL-positive segments in kidney organoids to detect potential axial functional differences suggestive of the existence of S1, S2, and S3-like PT segments (30, 31).

Endocrine functions.

The PT also plays an important endocrine role through its response to parathyroid hormone (PTH) and the production of active vitamin D in the PT cells in mammalian kidneys. Kidney organoids have been reported to express the PTH receptor PTH1R and the 1 α-hydroxylase CYP27B1, which catalyzes the synthesis of active vitamin D (38). The expression of both increases with organoid maturation, and 1,25-vitamin D is produced and accumulates in culture media in response to PTH, consistent with functional calcium metabolism in kidney organoids (39).

Loop of Henle-Distal Nephron

Structure in kidney organoids.

A second tubular epithelium is found in kidney organoids, with cells expressing cadherin-1 (CDH1). In human kidneys, CDH1 is expressed in the loop of Henle (LOH), the distal convoluted tubule, the connecting tubule, and the collecting duct (18). Double positive (LTL and CDH1) segments are occasionally observed (13), which appear to correspond to cycling proximal tubular cells (represented by enrichment of cell cycle genes) in healthy human kidneys according to the single-cell human kidney atlas generated by Human Biomolecular Atlas Program (HuBMAP) and Kidney Precision Medicine Project (KPMP) (Fig. 1, E and F) (40, 41). Although discernable morphological differences allow renal physiologists to identify and microdissect discrete segments of the mammalian kidney under a binocular loupe (6) or by light microscopy, specific features have not been assigned thus far to tubules in kidney organoids. However, single-cell gene expression profiling revealed cell identities that correspond to each segment, similar to human data (4143). For instance, the LOH cluster within kidney organoids expressed SLC12A1, which encodes NKCC2 and marks distal straight tubules. Interestingly, developmental genes such as dickkopf WNT signaling pathway inhibitor 1 (DKK1) were also coexpressed in this segment, suggesting an immature state (43).

Water and electrolyte handling.

The lack of functional homogeneity of the CDH1-positive segment makes it challenging to assess as a whole. In the fully differentiated mammalian kidney, the mature LOH is subdivided into a thin descending [descending thin limb (DTL)]], ascending thin (ATL), and thick ascending limb (TAL), arranged in a specific hairpin shape. Together with this structural arrangement, the differential permeability to water via aquaporin (AQP)1 expression in the DTL and to sodium via NKCC2 expression in the TAL, allows the establishment of the axial osmotic gradient necessary for urine concentration by the countercurrent multiplication process (44). The TAL also accounts for the reabsorption of ∼20% of the filtered load of sodium and is the major site of calcium and magnesium paracellular reabsorption as well (6).

Still in the mature mammalian kidney, the LOH is followed by a segment called the distal convoluted tubule (DCT) which, although short, plays a major role in sodium balance hence extracellular volume homeostasis, through fine-tuning of the activity of the apical sodium-chloride cotransporter (NCC) (45). Finally, two consecutive segments, namely the connecting tubule (CNT), and immediately downstream, the collecting duct (CD) display very similar cellular composition but interestingly originate from two different embryological progenitors, respectively, the metanephric mesenchyme and the ureteric bud. However, there may be contributions of resident CD cell types from both the lineages in some parts of the CNT (46). The CNT and CD are crucial for the final fine-tuning of ion and water balance. They consist of two major cell populations (47). Principal cells (PC) mediate sodium reabsorption via the apical epithelial Na+ channel (ENaC), potassium secretion via the renal outer medullary K+ (ROMK) channel, and water reabsorption via AQP2. Adjacent intercalated cells (IC) are further subdivided into type A (IC-A) and type B (IC-B) cells, predominantly involved in acid and bicarbonate secretion, respectively, but also contributing to net urinary potassium excretion via the large-conductance (BK) channel (48, 49). IC-A has an apical H+-ATPase and a basolateral chloride/bicarbonate exchanger (AE1), and IC-B has a basolateral H+-ATPase and an apical chloride/bicarbonate exchanger (pendrin) (6).

The majority of these transport proteins have been identified in kidney organoids at the mRNA/protein level in several reports (5052), including those focused on the maturation of ureteric bud-derived organoids (51, 5355). It is only recently that the function of some of them has been demonstrated. Montalbetti et al. (56) confirmed the expression of ENaC, ROMK, and BK channels in kidney organoids and characterized their electrophysiological signatures by patch-clamp analyses. Shi et al. (57) reseeded their ureteric bud-derived CD kidney organoids onto a two-dimensional (2-D) epithelial model allowing them to observe an amiloride-sensitive transepithelial voltage and current suggestive of an effective ENaC-mediated sodium transport. Despite the identification of cell identities corresponding to some of the distal nephron segments, the anatomic organization of LOH in a discrete cortico-medullary axis has not yet been achieved in kidney organoids. Moreover, as the distal segments are a small fraction of the tubular epithelial cells found in the kidney organoids, their study and functionality are inevitably constrained by their limited quantity. New differentiation strategies are thus warranted to broaden the fraction of these cell types while maintaining the more proximal segments of the nephron.

Interstitium

Structure in kidney organoids.

Kidney organoids contain mesenchyme-derived stroma-like cells as demonstrated by their nuclear MEIS1 expression (58, 59) and PDGFR membrane and cytoplasmic expression (Fig. 1G) (22). These cells are found embedded within the spaces between the aforementioned parts of the nephrons and in the periphery of the organoids. Their number tends to increase in later-stage organoids (60). The distinct zonation observed in stromal cells in rodent kidneys has not yet been reported in kidney organoids (61).

Endocrine functions.

Interstitial cells include fibroblast-like cells, pericytes, and mesangial cells (62). Their two main endocrine functions are the production of renin in the juxta-glomerular apparatus and erythropoietin (EPO) throughout the cortex and the outer medulla in mammalian kidneys. In this respect, Shankar et al. (23) observed the production of renin by kidney organoids, which was further stimulated in vitro by cAMP. The expression of renin was more pronounced in a pericyte subset cluster by single-cell sequencing. Regarding the response to the renin-angiotensin system, the expression of the angiotensin II type 1 receptor (AT1R) and the angiotensin II type 2 receptor (AT2R) appear to be sequential, the former being strongly expressed in the early stages of differentiation, whereas the latter peaking at day 12 after initiation of induced pluripotent stem cell (iPSC) differentiation (63). In addition, treatment with angiotensin II during the middle phase of differentiation seems to increase the expression of podocyte markers such as WT1 and podocalyxin, suggestive of improved maturation (63). Finally, although no studies have shown kidney organoids produce functional EPO to date, EPO-producing cells have been engineered from human-induced pluripotent stem cells that respond to hypoxia (64).

Vasculature

Structure in kidney organoids.

When cultured under static conditions, kidney organoids contain cells expressing endothelial markers such as platelet and endothelial cell adhesion molecule (PECAM)-1 (CD31) located in the interstitium (Fig. 1G). Although they generally do not penetrate the glomeruli, these cells are organized in capillary-like 3-D networks without a distinct lumen (16). Thus current organoid protocols need substantial improvement in achieving vascular cell type and functional diversity.

ENHANCING KIDNEY ORGANOID MATURATION: THE KEY TO IMPROVING FUNCTION?

Response to Shear Stress

As kidneys receive 25% of the cardiac output, renal cells are submitted to continuous flow of decreasing rate along with vascular branching. Vascular flow is critical to deliver vital nutrients and oxygen to cells but also confers a direct effect on cellular homeostasis (65). Luminal flow rate subjects epithelial cells in the discrete nephron segments to hydrodynamic forces (66). Fluid shear stress has been shown to play an essential role in organ and vascular maturation during embryogenesis and angiogenesis (6769), partly mediated by calcium signaling (7072). This observation along with findings that vasculature facilitates the formation of the liver bud from stem cell-derived multiple lineages (73), led to the investigation of the role of superfusate flow on kidney organoid differentiation and maturation (Fig. 3A). Kidney organoid superfusion led first to an increase in vascular density within the organoids (Fig. 3B), second to trigger migration of endothelial cells into the developing renal corpuscle, and third to enhanced tubular maturation as demonstrated by the increased expression of specific PT transporters (74). Recently, Aceves et al. (76) confirmed the observations of an upregulation of drug transporters induced by fluid flow and cisplatin-induced nephrotoxicity in kidney organoids (14). Their study demonstrated the basolateral OCT2-dependent uptake of cisplatin in an organ-on-a-chip model using PT-like epithelium isolated from kidney organioids, as previously described (76).

Figure 3.

Figure 3.

Flow-induced vascularization and mechanosensing biological processes. A: an illustration of the kidney organoid-on-chip model. B: immunostaining displaying vascularized kidney organoids under flow. C and D: differentially expressed genes in kidney organoids cultured under flow and their GO terms implicated in mechanosensing signals. The images of A and B were taken from Homan et al. (74) and C and D were from Hiratsuka et al. (75). GPCR, G protein-coupled receptor; ROS, reactive oxygen species; TGF, transforming growth factor.

The mechanisms by which shear stress accelerates kidney organoid function are suggested by transcriptomic analyses and are currently under investigation (Fig. 3, C and D) (75). Although increases in superfusate flow may stimulate differentiation by continuously removing morphogen inhibitors and/or ensuring a steady supply of essential nutrients, other flow-dependent physical cues may be at play. Sensing of luminal fluid shear stress in the fully differentiated proximal tubule has been attributed to the mechanical perturbation of brush border microvilli that are made of actin filament bundles (66). In the distal nephron, primary cilia have been proposed as key mechanosensors (77). An interesting finding is that the mutations in primary ciliary proteins can result in the loss of epithelial polarity, disrupted proliferation, and the formation of cysts, including in kidney organoids (78). This pathophysiological process was validated by inducing cyst formation upon the initiation of fluid flow in a kidney organoid model of autosomal recessive polycystic kidney disease. Furthermore, this process was linked to mechanosensory signals related to RAC1 and FOS (75).

Other factors mediate mechanosensation as well. Luminal flow-induced transient increases in intracellular calcium concentration have been identified in both principal and intercalated cells in acutely deciliated cortical CDs (CCDs) of the mature kidney, albeit lacking the typical immediate high-amplitude spike proposed to be secondary to inositol (1,4,5)-trisphosphate (IP3)-mediated release (66, 79, 80). In fact, recent studies have revealed the presence of basolateral PIEZO1 channels in the mammalian kidney (81) as well as in isolated organoid tubules (82). Specifically, single-organoid tubules subject to an increase in luminal or basolateral (superfusate) flow exhibited a prompt increase in intracellular calcium concentration with the response to luminal flow increasing with advancing days of organoid culture. This maturation of Ca2+ mobilization was associated with a gradual increase in PIEZO1 channel expression in the cell membranes over time during long-term organoid culture (82). Note that Piezo1 is expressed in endothelial cells of developing blood vessels in mice and has been shown to be required for proper vascular development during gestation (83).

Response to In Vivo Transplantation

Our ability to reproduce a physiological environment in vitro, needed for optimal viability and health of engineered organs, is still limited. An integrated approach with specific in vivo assays is needed to demonstrate functional maturity of kidney organoids and to optimize their maturation. It may also show sustainability by transplanting them into immunodeficient animals to prevent rejection (84). Vascular engraftment and fusion with the host along with accelerated maturation and global vascularization have been shown in several reports, as well as effective dextran uptake by the transplanted organoids (21, 2328). Li et al. (85) have also shown significant creatinine accumulation in kidney organoid cysts in vivo, a finding that might be suggestive of effective glomerular filtration and/or tubular fluid concentration. Endocrine functions might also be improved following transplantation, as highlighted by the upregulation of renin production in transplanted kidney organoids (23, 86).

Recent Advances in the Ureteric Bud, Vascular and Stroma Differentiation

One strategy that might enhance the functional maturation of kidney organoids is to integrate nephron progenitor-derived and ureteric bud-derived organoids. This approach has been reported to create an architecture closer to mammalian kidneys (51, 55, 87, 88). Stromal cells play an important role in kidney development as well (61, 89, 90). Recently, Tanigawa et al. (86) showed, utilizing the latter approach, that adding stromal progenitors further improves organoid architecture and specialized stromal cell differentiation. These integrated approaches of incorporating and optimizing different lineages hold promise to make better organized and mature organoids with a remaining challenge of creating a contiguous tubular system of urine flow.

RESOURCES AND DATA FOR iPSC AND KIDNEY ORGANOID RESEARCH

Genetically engineered induced pluripotent stem cell lines (iPSCs) are being extensively utilized in the advancement of kidney organoid research. These iPSC lines, both control (parent) and reporter cell lines specifically designed to label major nephron cell types of proximal, distal, and collecting tubules, are being generated and used for various purposes. These include optimizing differentiation protocols and conducting biological experiments, as mentioned in the preceding sections. To ensure the quality and standardization of iPSC lines for kidney research, the ReBuilding a Kidney (RBK) consortium has taken the initiative. They have established a framework for authenticating iPSC lines, which involves validating the targeted integration of reporters, assessing genetic and chromosomal integrity, and confirming the ability to form kidney organoids. As part of this initiative, the RBK has created a central iPSC repository (https://www.atlas-d2k.org/resources/cell-lines/), from which investigators can request both parent and reporter cell lines (Table 1). Currently, several iPSC lines are already available (91). In collaboration with the ATLAS-D2K Center (data center for RBK and GUDMAP) and the Washington University Pediatric Center of Excellence in Nephrology (https://pcen.wustl.edu/), a new development enables the distribution of each vial of these cell lines to researchers free of charge (Table 1). This collaborative effort establishes an infrastructure that will continue to expand and incorporate new iPSC lines donated by investigators. It aims to accelerate research efforts in kidney organoids and regenerative medicine.

Table 1.

Resources for human kidney organoids

Resource Name Resource Type Website
Washington University Human iPS Core iPS cells https://pcen.wustl.edu
Atlas D2K center Cell line resources, Protocols, Functional assays handbook, Omics data https://www.atlas-d2k.org/
KPMP Kidney Tissue Atlas Single-cell transcriptome data (human kidney) https://atlas.kpmp.org/
HuBMAP Spatial multiomic maps (healthy human organs) https://portal.hubmapconsortium.org/
CellxGene by CZI Atlas of healthy and injured cell states and niches (human kidney) https://cellxgene.cziscience.com/
Kidney Interactive Transcriptomics Single-cell transcriptome data (human kidney Mouse kidney organoids) https://humphreyslab.com/SingleCell/
ENCODE study Functional genome data (human kidney organoids) https://www.encodeproject.org/
Gene Expression Omnibus Single-cell and bulk transcriptome data (human kidney organoids, human fetal and adult kidneys) https://www.ncbi.nlm.nih.gov/geo/ (e.g., GSE164647)

HuBMAP, Human Biomolecular Atlas Program; KPMP, Kidney Precision Medicine Project.

Importantly, the provision of standardized resources and protocols will enhance quality control and reduce technical variability when working with highly sophisticated procedures such as the creation of kidney organoids. In this perspective, there is rapid progress in generating single-cell and spatial multiomic datasets from the human kidney that have begun to be publicly available including the Kidney Tissue Atlas KPMP (41), the Kidney Interactive Transcriptomics (KIT) datasets (92), the ENCODE study (93), HuBMAP (94), or the NCBI Gene Expression Omnibus data set of functional genomics (95), originating from human kidney tissue as well as from kidney organoids. These resources, along with the Handbook of Functional Assays, offer promising approaches to help inform improved organoid maturation and functionality (Table 1).

CHALLENGES AND FUTURE DIRECTION

Kidney organoids offer unparalleled prospects for exploring renal physiology and diseases in human cells ex vivo, representing a groundbreaking avenue for scientific inquiry. Nonetheless, there exist notable obstacles that demand immediate attention and resolution. Ethical considerations necessary to guide responsible organoid use have been summarized by previous reviews (96, 97). Ethical issues are mainly divided according to their use in research or clinics, the latter being still a distant prospect in the field of kidney organoids. Regarding their use in research, the first issue is the use of human embryonic stem cells (hESCs). To obtain hESCs, the first step involves the destruction of a 5-day-old preimplantation embryo (blastula stage), which raises the debate on the moral status of the embryo. The alternative use of human-derived iPSCs is not devoid of ethical concerns either. Researchers thus also need ethical guidance to ensure the responsible use of these resources, including donor’s informed consent requirement, and the use of approved repositories (98).

Foremost among the technical and scientific challenges is the issue of batch variations in kidney organoid differentiation, as elucidated by Phipson et al. (99). The maintenance of human pluripotent stem cells (hPSCs) necessitates vigilant monitoring and careful passaging, precisely timed to initiate cellular differentiation effectively. To achieve successful organoid differentiation, multiple intricate steps must be undertaken, incorporating an assortment of multiple growth factors. Consequently, this process entails labor-intensive daily feeding and the meticulous administration of various growth factors and small molecules. These technical complexities significantly contribute to the intricacy of organoid research, causing unpredictable batch variations. This inherent nature of organoid research poses a considerable challenge when striving to obtain reproducible outcomes across disparate laboratories. Moreover, the development of multiple differentiation protocols aimed at generating kidney organoids from hPSCs further adds to the complexity of the field. As a result, it becomes crucial to establish clear and standardized metrics for successful organoid differentiation.

The advancements achieved through the RBK activities have propelled the development and functional exploration of kidney organoids to a significant extent. Consequently, we are now at a stage where it is both appropriate and necessary to establish key metrics that define successful differentiation. These metrics serve as crucial benchmarks, ensuring that kidney organoids possess the essential cellular components required for accurate representation and study of nephrons and interstitial cells (Fig. 4). To meet these metrics, kidney organoids must comprise the fundamental cellular constituents of nephrons, such as podocytes, proximal tubules, LOHs, and distal nephrons (including distal convoluted tubules and connecting tubules). Importantly, the segments need to be arranged in a contiguous manner. Another important aspect of the cellular arrangement is the establishment of cellular polarity, with distinct luminal and basal sides, which is essential for functional studies and proper modeling of kidney physiology. In addition, interstitial cells, encompassing stromal cells and endothelial cells, must be present within the kidney organoids. Their inclusion is paramount to emulate the microenvironment of the kidney accurately and enable comprehensive investigations of cellular interactions and signaling pathways within the organoid system. Moreover, for the organoids to be effective tools in toxicity studies and pharmacological research, the proximal tubules must express key drug transporters, such as OCT2, OAT1/3, and MDR1. These transporters play a crucial role in drug metabolism and elimination, making their presence essential for the accurate evaluation of drug responses and toxicity assessments. Although progress has been made in defining these metrics, there remains a need for further studies to establish the specific benchmarks for LOHs and distal nephrons. By conducting these studies, we will be able to refine our understanding of these critical components, ensuring that kidney organoids encompass a comprehensive representation of the nephron structure and function.

Figure 4.

Figure 4.

Key metrics for successful differentiation of kidney organoids. At days 79 of differentiation, the nephron progenitor cells (NPC) express SIX2, as shown by the immunostaining in two-dimensional (2-D) cultures. Matured three-dimensional (3-D) organoids must comprise podocyte clusters, proximal tubular epithelial cells, and distal tubular epithelial cells, as evidenced by podocalyxin (PODXL), Lotus tetragonolobus lectin (LTL), and cadherin 1 (CDH1) staining, respectively. They also bear CD31-positive endothelial cells and MEIS-positive interstitial stromal cells. Cellular polarity must be verified, with apical staining of PODXL within the podocyte clusters, conversely to nephrin (NPHS1) and synaptopodin (SYNPO) basal staining, and luminal staining of MDR1 in the proximal tubular epithelial cells.

CONCLUSIONS: TOWARD THE PRODUCTION AND EXPULSION OF URINE

The exploration of kidney organoid functionality paves the way for this highly dynamic research field to advance to the next level. Confirmation of physiological function utilizing optimized and easily performed functional assays is necessary and should be considered as quality control of kidney organoids. In this perspective, the framework when studying kidney organoid function is summarized in Table 2. Progress is still needed to optimize/enhance/refine organoid architecture, structure, and functional maturation of these tissues, and many challenges remain. The physiological relevance of kidney organoids is critical in justifying their use as a relevant experimental model of disease. Translation of knowledge gained from the rigorous morphological and functional analyses of maturing organoids promises to provide additional insight into the structure and function of the metanephric kidney in development, health, and disease. A vital step toward achieving physical and functional maturity in kidney organoids involves understanding cell type diversity, spatial relationships, and gene expression profiles across different stages of life, ranging from pediatric to adult. Various atlas efforts are underway to delineate these aspects. This knowledge will serve as a crucial roadmap for assessing the degree of maturity achieved in kidney organoids and for engineering functional subsegments of the nephron. Ultimately, it is anticipated that these efforts will enable the production of engineered functional kidneys for transplantation.

Table 2.

Practice points to assess kidney organoid function and future directions

Practice Points Research Agenda
Assess the structure
• Quality control of the organoids
• Staining of the nephron segments
Improve the segment identification
• Identification of the different parts of the distal nephron, Improve the architecture
• Developmental insight
Assess the function
• Live imaging of epithelial transport
• Transepithelial voltage and current
• Microperfusion of microdissected tubules
• Patch-clamp analysis of specific transporters
Improve the tools for glomerular filtration and tubular secretion and reabsorption assessment
• in vitro with live organoid imaging
• in vivo on experimental models
Assess the maturity
• Gene expression profiles: developmental vs. differentiated genes.
Enhance the maturation
• Bioengineered tools
• Flow-enhanced maturation
• In vivo maturation
• Improved differentiation methods

GRANTS

This work was supported by National Institutes of Health (NIH) Award DP2EB029388/DK133821 (to R.M.), NIH Grants R01DK038470 (to L.M.S), R01DK129285 (to L.M.S), UC2DK126023 (to L.M.S. and R.M.), U01DK114933 (to S.J.), U54DK134301 (to S.J.), P50DK133943 (to S.J.), U24DK135157 (to S.J.), U01EB028899/DK127587 (to R.M.), and R21DK129909 (to R.M.), the French National Research Agency ANR-22-CE14-0077-01, the Monahan Foundation in collaboration with the Fulbright program, and the Servier Institute (to N.T.).

DISCLOSURES

R.M. is an inventor on a patent related to this work filed by the President and Fellows of Harvard College and Mass General Brigham (PCT/US2018/036677). R.M. holds a stock option in Trestle Biotherapeutics. None of the other authors has any conflicts of interest, financial or otherwise, to disclose.

AUTHOR CONTRIBUTIONS

N.T., L.M.S., S.J., and R.M. conceived and designed conceived and designed the review; N.T. N.T. wrote the first draft of the manuscript; N.T. and R.M. analyzed data; N.T. and R.M. prepared figures; N.T., L.M.S., S.J., and R.M. drafted manuscript; N.T., L.M.S., S.J., and R.M. edited and revised manuscript; N.T., L.M.S., S.J., and R.M. approved final version of manuscript.

ACKNOWLEDGMENTS

We thank RBK members for the insightful discussion about kidney organoids and their functional assessment and Dr. Haruka Oishi and Ken Hiratsuka for kidney organoid images. BioRender was used for the design of the figures.

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