Kidney Organoids Research Articles

Reduced guanidinoacetate in plasma of patients with autosomal dominant Fanconi syndrome due to heterozygous P341L GATM variant and study of organoids towards treatment

AbstractAutosomal dominant Fanconi syndrome due to a GATM variant (GATM‐FS), causes accumulation of misfolded arginine‐glycine amidinotransferase (AGAT) in proximal renal tubules leading to cellular injury. GATM‐FS presents during childhood and progresses to end‐stage kidney disease (ESKD) in adults. We study creatine metabolism in two individuals of unrelated families with a known GATM variant and the effect of creatine supplementation in kidney organoids. Plasma and urine metabolites were measured by mass spectrometry. Brain creatine was assessed by magnetic resonance spectroscopy (MRS). Guanidinoacetate (GAA) synthesis by the AGAT mutant was measured in patient‐derived immortalized lymphocytes using stable isotopes of arginine and glycine. The effect of creatine on GATM expression was assessed in human kidney cells and organoids. Several family members from two unrelated families were diagnosed with Fanconi syndrome and had the c.1022C>T (p. P341L) variant in GATM. Two affected individuals in both families had moderately reduced plasma GAA levels. In comparison to wild‐type cells, GAA synthesis by patient‐derived GATMP341L+/− lymphoblastoid cell lines (LCL) was reduced, but not absent as in GATM cells from a patient with creatine deficiency syndrome. In vitro studies on human kidney organoids revealed reduced AGAT expression after treatment with creatine. Finally, we showed in one patient that creatine supplementation (5 g daily) substantially increased plasma creatine levels. We report low plasma and urine GAA in patients with autosomal dominant GATM‐FS and show that creatine downregulates AGAT in human kidney cells.

Efficient proximal tubule-on-chip model from hiPSC-derived kidney organoids for functional analysis of renal transporters

Renal transporters play critical roles in predicting potential drug-drug interactions. However, current invitro models often fail to adequately express these transporters, particularly solute carrier proteins, including organic anion transporters (OAT1/3), and organic cation transporter 2 (OCT2). Here, we developed a hiPSC-derived kidney organoids-based proximal tubule-on-chip (OPTC) model that emulates invivo renal physiology to assess transporter function. Compared to chips based on immortalized cells, OPTC derived from the two most commonly used differentiation protocols exhibited significant improvement in expression level and polarity of OAT1/3 and OCT2. Hence, the OPTC demonstrates enhanced functionality in efflux and uptake assessments, and nephrotoxicity. Furthermore, these functionalities are diminished upon adding inhibitors during substrate-inhibitor interactions, which were closer to invivo observations. Overall, these results support that OPTC can reliably assess the role of renal transporters in drug transport and nephrotoxicity, paving the way for personalized models to assess renal transport and disease modeling.

Generating kidney organoids based on developmental nephrology

Over the past decade, the induction protocols for the two types of kidney organoids (nephron organoids and ureteric bud organoids) from pluripotent stem cells (PSCs) have been established based on the knowledge gained in developmental nephrology. Kidney organoids are now used for disease modeling and drug screening, but they also have potential as tools for clinical transplantation therapy. One of the options to achieve this goal would be to assemble multiple renal progenitor cells (nephron progenitor, ureteric bud, stromal progenitor) to reproduce the organotypic kidney structure from PSCs. At least from mouse PSCs, all the three progenitors have been induced and assembled into such “higher order” kidney organoids. We will provide an overview of the developmental nephrology required for the induction of renal progenitors and discuss recent advances and remaining challenges of kidney organoids for clinical transplantation therapy.

The Impairment of Endothelial Autophagy Accelerates Renal Senescence by Ferroptosis and NLRP3 Inflammasome Signaling Pathways with the Disruption of Endothelial Barrier.

Autophagy is a cellular process that degrades damaged cytoplasmic components and regulates cell death. The homeostasis of endothelial cells (ECs) is crucial for the preservation of glomerular structure and function in aging. Here, we investigated the precise mechanisms of endothelial autophagy in renal aging. The genetic deletion of Atg7 in the ECs of Atg7flox/flox;Tie2-Cre mice accelerated aging-related glomerulopathy and tubulointerstitial fibrosis. The EC-specific Atg7 deletion in aging mice induced the detachment of EC with the disruption of glomerular basement membrane (GBM) assembly and increased podocyte loss resulting in microalbuminuria. A Transwell co-culture system of ECs and kidney organoids showed that the iron and oxidative stress induce the disruption of the endothelial barrier and increase vascular permeability, which was accelerated by the inhibition of autophagy. This resulted in the leakage of iron through the endothelial barrier into kidney organoids and increased oxidative stress, which led to ferroptotic cell death. The ferritin accumulation was increased in the kidneys of the EC-specific Atg7-deficient aging mice and upregulated the NLRP3 inflammasome signaling pathway. The pharmacologic inhibition of ferroptosis with liproxstatin-1 recovered the disrupted endothelial barrier and reversed the decreased expression of GPX4, as well as NLRP3 and IL-1β, in endothelial autophagy-deficient aged mice, which attenuated aging-related renal injury including the apoptosis of renal cells, abnormal structures of GBM, and tubulointerstitial fibrosis. Our data showed that endothelial autophagy is essential for the maintenance of the endothelial barrier during renal aging and the impairment of endothelial autophagy accelerates renal senescence by ferroptosis and NLRP3 inflammasome signaling pathways. These processes may be attractive therapeutic targets to reduce cellular injury from renal aging.

Cryopreservation of human kidney organoids

Recent advances in stem cell research have led to the creation of organoids, miniature replicas of human organs, offering innovative avenues for studying diseases. Kidney organoids, with their ability to replicate complex renal structures, provide a novel platform for investigating kidney diseases and assessing drug efficacy, albeit hindered by labor-intensive generation and batch variations, highlighting the need for tailored cryopreservation methods to enable widespread utilization. Here, we evaluated cryopreservation strategies for kidney organoids by contrasting slow-freezing and vitrification methods. 118 kidney organoids were categorized into five conditions. Control organoids followed standard culture, while two slow-freezing groups used 10% DMSO (SF1) or commercial freezing media (SF2). Vitrification involved V1 (20% DMSO, 20% Ethylene Glycol with sucrose) and V2 (15% DMSO, 15% Ethylene Glycol). Assessment of viability, functionality, and structural integrity post-thawing revealed notable differences. Vitrification, particularly V1, exhibited superior viability (91% for V1, 26% for V2, 79% for SF1, and 83% for SF2 compared to 99.4% in controls). 3D imaging highlighted distinct nephron segments among groups, emphasizing V1’s efficacy in preserving both podocytes and tubules in kidney organoids. Cisplatin-induced injury revealed a significant reduction in regenerative capacities in organoids cryopreserved by flow-freezing methods, while the V1 method did not show statistical significance compared to the unfrozen controls. This study underscores vitrification, especially with high concentrations of cryoprotectants, as an effective approach for maintaining kidney organoid viability and structure during cryopreservation, offering practical approaches for kidney organoid research.

Advancements in Research on Genetic Kidney Diseases Using Human-Induced Pluripotent Stem Cell-Derived Kidney Organoids.

Genetic or hereditary kidney disease stands as a pivotal cause of chronic kidney disease (CKD). The proliferation and widespread utilization of DNA testing in clinical settings have notably eased the diagnosis of genetic kidney diseases, which were once elusive but are now increasingly identified in cases previously deemed CKD of unknown etiology. However, despite these diagnostic strides, research into disease pathogenesis and novel drug development faces significant hurdles, chiefly due to the dearth of appropriate animal models and the challenges posed by limited patient cohorts in clinical studies. Conversely, the advent and utilization of human-induced pluripotent stem cells (hiPSCs) offer a promising avenue for genetic kidney disease research. Particularly, the development of hiPSC-derived kidney organoid systems presents a novel platform for investigating various forms of genetic kidney diseases. Moreover, the integration of the CRISPR/Cas9 technique into this system holds immense potential for efficient research on genetic kidney diseases. This review aims to explore the applications of in vitro kidney organoids generated from hiPSCs in the study of diverse genetic kidney diseases. Additionally, it will delve into the limitations of this research platform and outline future perspectives for advancing research in this crucial area.

Stepwise developmental mimicry generates proximal-biased kidney organoids.

The kidney maintains body fluid homeostasis by reabsorbing essential compounds and excreting waste. Proximal tubule cells, crucial for renal reabsorption of a range of sugars, ions, and amino acids, are highly susceptible to damage, leading to pathologies necessitating dialysis and kidney transplants. While human pluripotent stem cell-derived kidney organoids are used for modeling renal development, disease, and injury, the formation of proximal nephron cells in these 3D structures is incomplete. Here, we describe how to drive the development of proximal tubule precursors in kidney organoids by following a blueprint of in vivo human nephrogenesis. Transient manipulation of the PI3K signaling pathway activates Notch signaling in the early nephron and drives nephrons toward a proximal precursor state. These "proximal-biased" (PB) organoid nephrons proceed to generate proximal nephron precursor cells. Single-cell transcriptional analyses across the organoid nephron differentiation, comparing control and PB types, confirm the requirement of transient Notch signaling for proximal development. Indicative of functional maturity, PB organoids demonstrate dextran and albumin uptake, akin to in vivo proximal tubules. Moreover, PB organoids are highly sensitive to nephrotoxic agents, display an injury response, and drive expression of HAVCR1 / KIM1 , an early proximal-specific marker of kidney injury. Injured PB organoids show evidence of collapsed tubules, DNA damage, and upregulate the injury-response marker SOX9 . The PB organoid model therefore has functional relevance and potential for modeling mechanisms underpinning nephron injury. These advances improve the use of iPSC-derived kidney organoids as tools to understand developmental nephrology, model disease, test novel therapeutics, and for understanding human renal physiology.

Protocol for the Growth and Maturation of hiPSC-Derived Kidney Organoids using Mechanically Defined Hydrogels.

With recent advances in the reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs), gene editing technologies, and protocols for the directed differentiation of stem cells into heterogeneous tissues, iPSC-derived kidney organoids have emerged as a useful means to study processes of renal development and disease. Considerable advances guided by knowledge of fundamental renal developmental signaling pathways have been made with the use of exogenous morphogens to generate more robust kidney-like tissues in vitro. However, both biochemical and biophysical microenvironmental cues are major influences on tissue development and self-organization. In the context of engineering the biophysical aspects of the microenvironment, the use of hydrogel extracellular scaffolds for organoid studies has been gaining interest. Two families of hydrogels have recently been the subject of significant attention: self-assembling peptide hydrogels (SAPHs), which are fully synthetic and chemically defined, and gelatin methacryloyl (GelMA) hydrogels, which are semi-synthetic. Both can be used as support matrices for growing kidney organoids. Based on our recently published work, we highlight methods describing the generation of human iPSC (hiPSC)-derived kidney organoids and their maturation within SAPHs and GelMA hydrogels. We also detail protocols required for the characterization of such organoids using immunofluorescence imaging. Together, these protocols should enable the user to grow hiPSC-derived kidney organoids within hydrogels of this kind and evaluate the effects that the biophysical microenvironment provided by the hydrogels has on kidney organoid maturation. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Directed differentiation of human induced pluripotent stem cells (hiPSCs) into kidney organoids and maturation within mechanically tunable self-assembling peptide hydrogels (SAPHs) Alternate Protocol: Encapsulation of day 9 nephron progenitor aggregates in gelatin methacryloyl (GelMA) hydrogels. Support Protocol 1: Human induced pluripotent stem cell (hiPSC) culture. Support Protocol 2: Organoid fixation with paraformaldehyde (PFA) Basic Protocol 2: Whole-mount immunofluorescence imaging of kidney organoids. Basic Protocol 3: Immunofluorescence of organoid cryosections.

Patient-derived and gene-edited pluripotent stem cells lacking NPHP1 recapitulate juvenile nephronophthisis in abnormalities of primary cilia and renal cyst formation.

Juvenile nephronophthisis is an inherited renal ciliopathy with cystic kidney disease, renal fibrosis, and end-stage renal failure in children and young adults. Mutations in the NPHP1 gene encoding nephrocystin-1 protein have been identified as the most frequently responsible gene and cause the formation of cysts in the renal medulla. The molecular pathogenesis of juvenile nephronophthisis remains elusive, and no effective medicines to prevent end-stage renal failure exist even today. No human cellular models have been available yet. Here, we report a first disease model of juvenile nephronophthisis using patient-derived and gene-edited human induced pluripotent stem cells (hiPSCs) and kidney organoids derived from these hiPSCs. We established NPHP1-overexpressing hiPSCs from patient-derived hiPSCs and NPHP1-deficient hiPSCs from healthy donor hiPSCs. Comparing these series of hiPSCs, we found abnormalities in primary cilia associated with NPHP1 deficiency in hiPSCs. Kidney organoids generated from the hiPSCs lacking NPHP1 formed renal cysts frequently in suspension culture with constant rotation. This cyst formation in patient-derived kidney organoids was rescued by overexpression of NPHP1. Transcriptome analysis on these kidney organoids revealed that loss of NPHP1 caused lower expression of genes related to primary cilia in epithelial cells and higher expression of genes related to the cell cycle. These findings suggested the relationship between abnormality in primary cilia induced by NPHP1 loss and abnormal proliferative characteristics in the formation of renal cysts. These findings demonstrated that hiPSC-based systematic disease modeling of juvenile nephronophthisis contributed to elucidating the molecular pathogenesis and developing new therapies.

Postnatal renal tubule development: roles of tubular flow and flux.

Postnatal renal tubule development is critical to adult kidney function. Several postnatal changes regulate the differentiation and proliferation of renal tubular cells. Here, we review the literature and our efforts on thick ascending limb (TAL) development in Bartter syndrome (BS). Glomerular filtrate quickly increases after birth, imposing fluid shear stress and circumferential stretch on immature renal tubules. Recent studies showed that kidney organoids under flow (superfusion) have better development of tubular structures and the expression of cilia and solute transporters. These effects are likely mediated by mechanosensors, such as cilia and the piezo1 channel. Improved renal oxygenation and sodium pump-dependent active transport can stimulate mitochondrial respiration and biogenesis. The functional coupling between transport and mitochondria ensures ATP supply for energy-demanding reactions in tubular cells, including cell cycle progression and proliferation. We recently discovered that postnatal renal medulla maturation and TAL elongation are impaired in Clc-k2-deficient BS mice. Primary cultured Clc-k2-deficient TAL cells have G1-S transition and proliferation delay. These developmental defects could be part of the early pathogenesis of BS and worsen the phenotype. Understanding how tubular flow and transepithelial ion fluxes regulate renal tubule development may improve the treatment of congenital renal tubulopathies.

A perfusable, vascularized kidney organoid-on-chip model.

The ability to controllably perfuse kidney organoids would better recapitulate the native tissue microenvironment for applications ranging from drug testing to therapeutic use. Here, we report a perfusable, vascularized kidney organoid on chip model composed of two individually addressable channels embedded in an extracellular matrix (ECM). The channels are respectively seeded with kidney organoids and human umbilical vein endothelial cells (HUVECs) that form a confluent endothelium (macrovessel) During perfusion, endogenous endothelial cells present within the kidney organoids migrate through the ECM towards the macrovessel, where they form lumen-on-lumen anastomoses that are supported by stromal cells. Once micro-macrovessel integration is achieved, we introduced fluorescently labeled dextran of varying molecular weight and red blood cells into the macrovessel, which are transported through the microvascular network to the glomerular epithelia within the kidney organoids. Our approach for achieving controlled organoid perfusion opens new avenues for generating other organ-specific human tissues.&#xD.

Natural Hydrogels Support Kidney Organoid Generation and Promote In Vitro Angiogenesis.

To date, strategies aiming to modulate cell to extracellular matrix (ECM) interactions during organoid derivation remain largely unexplored. Here renal decellularized ECM (dECM) hydrogels are fabricated from porcine and human renal cortex as biomaterials to enrich cell-to-ECM crosstalk during the onset of kidney organoid differentiation from human pluripotent stem cells (hPSCs). Renal dECM-derived hydrogels are used in combination with hPSC-derived renal progenitor cells to define new approaches for 2Dand 3Dkidney organoid differentiation, demonstrating that in the presence of these biomaterials the resulting kidney organoids exhibit renal differentiation features and the formation of an endogenous vascular component. Based on these observations, a new method to produce kidney organoids with vascular-like structures is achieved through the assembly of hPSC-derived endothelial-like organoids with kidney organoids in 3D. Major readouts of kidney differentiation and renal cell morphology are assessed exploiting these culture platforms as new models of nephrogenesis. Overall, this work shows that exploiting cell-to-ECM interactions during the onset of kidney differentiation from hPSCs facilitates and optimizes current approaches for kidney organoid derivation thereby increasing the utility of these unique cell culture platforms for personalized medicine.

A genetically inducible endothelial niche enables vascularization of human kidney organoids with multilineage maturation and emergence of renin expressing cells

Vascularization plays a critical role in organ maturation and cell type development. Drug discovery, organ mimicry, and ultimately transplantation hinge on achieving robust vascularization of in vitro engineered organs. Here, focusing on human kidney organoids, we overcame this hurdle by combining a human induced pluripotent stem cell (iPSC) line containing an inducible ETS translocation variant 2 (ETV2) (a transcription factor playing a role in endothelial cell development) that directs endothelial differentiation in vitro, with a non-transgenic iPSC line in suspension organoid culture. The resulting human kidney organoids show extensive endothelialization with a cellular identity most closely related to human kidney endothelia. Endothelialized kidney organoids also show increased maturation of nephron structures, an associated fenestrated endothelium with de novo formation of glomerular and venous subtypes, and the emergence of drug-responsive renin expressing cells. The creation of an engineered vascular niche capable of improving kidney organoid maturation and cell type complexity is a significant step forward in the path to clinical translation. Thus, incorporation of an engineered endothelial niche into a previously published kidney organoid protocol allowed the orthogonal differentiation of endothelial and parenchymal cell types, demonstrating the potential for applicability to other basic and translational organoid studies.

Kidney Organoid Modeling of WT1 Mutations Reveals Key Regulatory Paths Underlying Podocyte Development.

Wilms tumor-1(WT1) is a crucial transcription factor that regulates podocyte development. However, the epigenomic mechanism underlying the function of WT1 during podocyte development has yet to be fully elucidated. Here, single-cell chromatin accessibilityand gene expressionmaps of foetal kidneys and kidney organoids are generated. Functional implications of WT1-targeted genes, which are crucial for the development of podocytes and the maintenance of their structure, including BMPER/PAX2/MAGI2 that regulates WNT signaling pathway, MYH9 that maintains actin filament organization and NPHS1 that modulates cell junction assembly are identified. To further illustrate the functional importance of WT1-mediated transcriptional regulation during podocyte development, cultured and implanted patient-derived kidney organoids derived from the Induced Pluripotent Stem Cell (iPSCs) of a patient with a heterozygous missense mutation in WT1 are generated. Results from single-cell RNA sequencing (scRNA-seq)and functional assays confirm that the WT1 mutation leads to delays in podocyte development and causes damage to cell structures, due to its failure to activate the targeting genes MAGI2, MYH9, and NPHS1. Notably, correcting the mutation in the patient iPSCs using CRISPR-Cas9 gene editing rescues the podocyte phenotype. Collectively, this work elucidates the WT1-related epigenomic landscape with respect to human podocyte development and identifies the disease-causing role of a WT1 mutation.

#930 Elevated glucose in kidney organoids induces epithelial detachment driven by proinflammatory cytokines

Abstract Background and Aims Diabetic kidney disease (DKD) is the leading cause of chronic kidney disease affecting 40% of diabetics. The pathogenesis of DKD is linked with inflammation, altered metabolism, and hemodynamic change but the relationship between these factors is not well understood. Proteinuria and albuminuria are common features, suggesting that impairments in the renal glomerular filtration barrier may be among the primary disease initiators. Limited DKD models are available, which has hampered our disease understanding and the ability to develop new therapies. Therefore, creating an in-vitro model that harbors multiple renal cell types organized in their natural complex arrangement becomes an attractive option to fill in this gap. Here, we conducted a systematic investigation of the effects of high glucose on kidney organoids, with the goal of understanding whether these cultures can reflect specific features relevant to DKD. Method To evaluate the effect of high glucose, kidney organoids were differentiated until maturation (day 21) in standard glucose and then switched for six days to concentrations of 5.5, 11, 33 and 100 mM. Then, to test the effect of proinflammatory cytokines on the injury phenotypes we switched organoids, at day 21, in control glucose condition (11 mM) ± TNFα or high glucose (33 mM) ± inhibitors (TNFα). Morphology was analysed via immunostaining, LDH and live/dead assay were used for toxicity, qPCR and western blotting to investigate differences in gene and protein expression, and mannose was used as an osmotic control. scRNA-seq of organoid in 11 vs 33 mM was conducted to gain insight into the molecular mechanism(s) contributing to the high glucose. Results Organoids remain well preserved in glucose up to 11 mM whereas organoids cultured in 33 and 100 mM showed a ∼30% reduction in overall organoid number between days 3 and 6. Moreover, in high glucose (33 and 100 mM), we observed an increase in the number of small, single PODXL+ cells surrounding the main organoid body, suggesting detachment from the podocyte clusters that normally form in organoids (Fig. A). To quantify it, we divided the total PODXL+ area into two sub-regions, classified as intact area and detached area, and calculated their ratio (Fig. B). Results indicated a 2-fold reduction of the intact vs detached area between 11 and 33 mM (p = 0.0011), suggesting that organoids undergo a spreading process under the influence of high glucose (Fig. C). Comparison of equimolar glucose vs mannose, live/dead and LDH assays identify a non-osmotic and non-apoptotic cause of the injury. ScRNA-seq analysis identified TNF-α as an upregulated pathway, which was confirmed with qPCR (p = 0.0131). Comparison of organoids at 11mM ± TNFα indicated that TNFα addition sensitizes podocytes to detachment at lower glucose levels, specifically, we identified a 4-fold reduction of the PODXL+ intact vs detached area at 11 mM ± TNFα (p < 0.0001), Fig. D. To further validate these findings and investigate possible therapeutic strategies, organoids were cultured with 33 mM glucose with TNF-α inhibitor and we identified a protective effect of the inhibitor which prevented the podocyte spreading phenotype at 33 mM glucose (Fig. E). Conclusion Our experiments identify an injury phenotype under high glucose concentrations, in kidney organoids, characterized by podocyte detachment from the main organoid body. Moreover, no osmotic or toxicity effect was observed. Importantly, this phenotype resembles disease features identified in diabetic patients, where podocyte loss and detachment from the glomerular basement membrane is associated with pathophysiology. We identified a correlation between the injury phenotype, characterized by epithelial detachment, and proinflammatory cytokine upregulation, which is driven by high glucose, and we have shown it can be targeted therapeutically. Therefore, this new experimental model in-vitro offers up new insights into the pathophysiology of diabetes and its complications, and represents a new platform to screen for therapeutics.

#1805 Renal progenitor cells isolated from urine of IgA nephropathy patients formed renal spheroids that can recapitulate the typical IgA1 deposition

Abstract Background and Aims Recent developments in stem cell biology have made it possible to create spheroids and kidney organoids from human and mouse pluripotent stem cells and renal stem cells. Spheroids are a suitable in vitro model for studies of drug studies and can transform into organoids whose rapidly developing field has proven useful in simulating nephrotoxicity, kidney disorders, and kidney development. However, to date in all studies the generation of organoids and tubuloids required the use of sophisticated and complex differentiation protocols involving external administration of several growth factors and needs compliance with a precise and specific timescale. Here we showed for the first time that human adult renal progenitor cells (ARPCs) can be isolated from urines of healthy subjects (HS) or IgA Nephropathy (IgAN) patients and can form spheroids generating very long tubule-like structures naturally, without the external use of chemokines or growth factors to artificially induce the process and recapitulating the IgA1 deposition that is the typical characteristic of the disease. Method Spheroids and tubule-like structures were characterized by whole-mounting and frozen section immunofluorescence (IF) assays and by Flow Cytometry Analysis using CD133, NanoG, Oct3/4, GATA-3, SSEA4, CD249, Aminopepidase N, ZO-1, Uromodulin, and Lotus Tetragonolobus Lectin (LTL) antibodies. ELISA was used to determine the renin secretion and CAM assays to determine ARPCs and ARPC-derived Spheroids angiogenic properties. PKH-26 labeling was used for their in vitro and in vivo tracking. Serum of HS and IgAN patients in culture with spheroids was used to recapitulate the disease and IgA1 deposits were measured by IF. Results ARPC-derived spheroids reflected the renal progenitor properties and recapitulated organoids generating very long tubule-like structures that expressed structural and functional renal tubule markers such as CD249, Aminopepidase N, ZO-1, Uromodulin, and LTL and in some cases shared many structural similarities with some regions of nephrons, such as the distal convoluted tubule, the loop of Henle and proximal convoluted tubules. Moreover, ARPC-derived spheroids secreted elevated levels of renin and, as the cellular counterpart, showed angiogenic properties. To study the proof of concept that ARPC-derived spheroids can be used to establish in vitro models recapitulating renal diseases, we generated IgAN patient-specific renal spheroids using the ARPC mixed cell population method. ARPC-derived spheroids of IgAN patients showed deposits of IgA1 already after four days of culture with patient serum (p = 0.004) and, with a different pattern, after 8 and 15 days (p = 0.0029 and p = 0.0012, respectively). The IgA1 deposition resembled that in the glomeruli of patients diagnosticated with IgAN. In contrast, no positive signal was detected in either IgAN spheroids in culture with FBS, or with inactivated IgAN serum, as well as in spheroids derived from urine of HS in culture with IgAN serum. Conclusion The ability of ARPCs to form spheroids and differentiate into tubular-like structures without the need for external chemokines or growth factors is a significant advancement in the field, even more considering that these cells can be isolated from the urine of patients. These findings open up new possibilities for the regenerative medicine in studying kidney development, disease mechanisms, and regenerative therapy development for renal disorders.

#1344 Molecular signatures of assembloids generated from stem cell derived nephron progenitors and ureteric bud cells as potential kidney organoid therapy

Abstract Background and Aims Novel therapies are needed to address the unmet clinical need of patients suffering from end stage kidney disease (ESKD) and cell therapy is an innovative strategy to deliver effective, curative treatment options to renal patients. There are several reports that have developed protocols to differentiate human pluripotent stem cells (hPSCs) into kidney-lineage progenitor derived organoid models and we have developed a robust and scalable method to produce kidney organoids. Method hPSCs were used to generate nephron progenitor cells (NPCs) and ureteric bud cells (UB) through previously described stem cell differentiation protocols. NPCs on day 9 of differentiation and UB on day 9 were combined for 2 days at 3:1 ratio respectively, to form ‘assembloids’ in-vitro and implanted in-vivo for 2 weeks. We used kidney capsule implantation in a NOD background mouse model and studied integration and phenotype using micro-CT and immunohistochemistry. Results RNA sequencing, flow cytometry and histology of NPCs, UB and assembloids in vitro shows broad representation of renal cell types by expression of markers representing several cell populations present in the developing nephron. We can currently show in-vitro molecular signatures of NPC derived organoids versus assembloids, with superiority of UB derived structures such as connecting tubules and intercalated cells. In-vivo histological examination of implanted assembloids showed maturation of human renal compartments such as collecting ducts, distal tubules as well as glomeruli-like structures. Micro-CT analysis showed integration with host and host derived endothelial vascularisation. Conclusion These observations demonstrate the use and differentiation potential of hPSCs and organoids in a pre-clinical setting towards more functional like kidney compartments by combining different progenitor populations. More work is needed evaluating these and other renal progenitor populations for future efforts to examine efficacy studies to develop regenerative therapies for ESKD.

#512 Two are better than one: a new combination treatment for cystinosis

Abstract Background and Aims Nephropathic cystinosis is a rare, lysosomal storage disorder resulting from mutations in the CTNS gene, encoding a cystine transporter. Disruption of this protein leads to the accumulation of cystine in cells and the formation of crystals that damage tissues and organs. Patients usually present around 6-months of age with failure to thrive, excessive thirst (polydipsia) and urination (polyuria) and Fanconi syndrome (excessive loss of important nutrients in the urine). The kidneys are seriously affected due to proximal tubule dysfunction and if left untreated this can lead to kidney failure by the age of 10. Patients receive the drug-based therapy cysteamine from the time of diagnosis. However, even with prolonged treatment, individuals may still advance to kidney failure, necessitating transplantation. As such, there is an urgent need for alternative treatments. In previous work using stem cell and kidney organoid models, we discovered that a drug combination of cysteamine and the mTOR inhibitor, Everolimus, can correct the cystinotic cell phenotype. To evaluate the therapeutic potential of this therapy in vivo, we generated a cystinotic rat model that faithfully recapitulates the human disease phenotype within 3–6 months as seen by: failure to gain weight, polydipsia and polyuria, cystine accumulation, Fanconi syndrome and kidney dysfunction. Using these Ctns knockout (KO) rats we performed pre-clinical testing to evaluate the therapeutic potential of Cysteamine/Everolimus combination treatment. Method Six-week-old male Ctns KO rats (n = 6) were dosed with either vehicle, Cysteamine-only, Everolimus-only or combination (Cysteamine and Everolimus) delivered via jelly pills for 6 months. Body weights were recorded weekly and blood and urine collected monthly for in-depth chemical analysis of Fanconi syndrome markers. At the end of the study, tissues were harvested for cystine measurements, immunohistochemistry and histology. Results Cysteamine monotreatment was efficacious in ameliorating the disease phenotype while Everolimus alone showed mixed results. However, combination treatment resulted in a superior reduction in tissue cystine levels, polydipsia, polyuria and a superior improvement in Fanconi syndrome markers, gross kidney morphology and histology compared to monotreatments. Specifically, in-depth urine analysis at 6 months showed significantly reduced levels of albumin, total protein and glucose in combination-treated animals compared to cysteamine monotreatment. Furthermore, the kidneys of combination-treated animals showed a greater amount of undamaged proximal renal tubules when compared to vehicle, cysteamine and Everolimus-treated kidneys along with a greater preservation of Lrp2/Megalin. Conclusion Overall the combination treatment resulted in greater preservation and protection of renal structures compared to monotreatments. These results demonstrate the potential of a Cysteamine/Everolimus dual therapy to improve the treatment of cystinosis.

Vascularization of kidney organoids: different strategies and perspectives

Kidney diseases such as glomerulopathy and nephron dysfunction are estimated to grow to more than 900 million cases by 2030, in 45% of which kidney transplantation will be required, representing a major challenge for biomedicine. A wealth of progress has been made to model human diseases using induced pluripotent stem cells (iPSCs) in vitro differentiated to a variety of organoids, including kidney organoids, and in developing various microfluidics-based organ-on-a-chip (OoC) systems based on them. With the combination of targeted gene editing capacities, relevant polymorphic genetic variants can be established in such organoid models to advance evidence-based medicine. However, the major drawback of the current organoid disease models is the lack of functional endothelial vasculature, which especially concerns the kidney, the function of which is strongly associated with blood flow. The design of novel medical devices using tissue engineering approaches such as kidney organoids is also strongly dependent on the understanding of the fundamental principles of nephrogenesis and the vascularization of organs and tissues. Developmental vascularization of the kidney has been an area of intense research for decades. However, there is still no consensus among researchers on how exactly the vascularization of the kidney occurs in normal and pathological conditions. This lack of consensus is partly due to the lack of an appropriate model system to study renal vascularization during nephrogenesis. In this review, we will describe recent progress in the areas of kidney vasculature development, kidney organoids in general and assembled on microfluidic devices in particular. We will focus on the in vitro vasculature of kidney organoids in microfluidic OoC model systems to study kidney diseases and on the perspectives of tissue engineering for the modeling of kidney diseases and the design of bioartificial medical devices. We also aim to summarize the information related to the key mechanisms of intercellular communication during nephrogenesis and the formation of the renal vasculature in an OoC setup.

Guidelines for Manufacturing and Application of Organoids: Kidney.

Recent advancements in organoid technology have led to a vigorous movement towards utilizing it as a substitute for animal experiments. Organoid technology offers versatile applications, particularly in toxicity testing of pharmaceuticals or chemical substances. However, for the practical use in toxicity testing, minimal guidance is required to ensure reliability and relevance. This paper aims to provide minimal guidelines for practical uses of kidney organoids derived from human pluripotent stem cells as a toxicity evaluation model in vitro.