The renin-angiotensin system (RAS) is essential for normal kidney development. Dysregulation of the RAS during embryogenesis can result in kidney abnormalities. To explore how angiotensin type 1 receptor (AT1R) signaling modulates nephron progenitor (NP) fate specification, we used induced pluripotent stem cell (iPSC) derived human kidney organoids treated with angiotensin II (Ang II) or the AT1R blocker losartan during differentiation. Ang II promoted NP proliferation and differentiation preferentially toward a podocyte fate, depleted the podocyte precursor population, and accelerated glomerular maturation. By contrast, losartan expanded the podocyte precursor population, delayed podocyte differentiation, and regressed the transcriptional signature to a more immature fetal state. Overall, using various in silico approaches with validation by RNAscope, we identified a role for AT1R signaling in regulating NP fate during nephrogenesis in kidney organoids. Our work supports the use of RAS modulators to improve organoid maturation and suggests that RAS may be a determinant of nephron endowment in vivo.

CRISPR-based genetically modified scaffold-free biomaterials, including extracellular vehicles, cell sheets, cell aggregates, organoids and organs, have attracted significant attention in the fields of regenerative medicine and tissue engineering in recent years. With a wide range of applications in gene therapy, modeling disease, tissue regeneration, organ xenotransplantation, modeling organogenesis as well as gene and drug screening, they are at a critical juncture from clinical trials to therapeutic applications. Xenografts have already been tested on non-human primates and humans. However, we have to admit that a series of obstacles still need to be addressed, such as immune response, viral infection, off-target effects, difficulty in mass production, and ethical issues. Therefore, future research should pay more attention to improving their safety, accuracy of gene editing, flexibility of production, and ethical rationality. This review summarizes various types of CRISPR-based genetically modified scaffold-free biomaterials, including their preparation procedures, applications, and possible improvements.

Human pluripotent stem cells hold inherent properties, allowing researchers to recapitulate key morphogenetic processes. These characteristics, coupled with bioengineering techniques, have led to the definition of early procedures to derive organ-like cell cultures, the so-called organoids. With regard to kidney organoids, challenges stand ahead, such as the need to enhance cellular composition, maturation, and function to that found in the native organ. To this end, the kidney organoid field has begun to nourish from innovative engineering approaches aiming to gain control on the externally imposed biochemical and biophysical cues. In this review, we first introduce how previous research in kidney development and human pluripotent stem cells has informed the establishment of current kidney organoid procedures. We then discuss recent engineering approaches to guide kidney organoid self-organization, differentiation, and maturation. In addition, we present current strategies to engineer vascularization and promote in vivo-like physiological microenvironments as potential solutions to increase kidney organoid lifespan and functionality. We finally emphasize how working at the cusp of cell mechanics and computational biology will set the ground for successful translational applications of kidney organoids.

Organoid technology has significantly transformed biomedical research by providing exceptional prospects for modeling human tissues and disorders in a laboratory setting. It has significant potential for understanding the intricate relationship between genetic mutations, cellular phenotypes, and disease pathology, especially in the field of genetic diseases. The intersection of organoid technology and genetic research offers great promise for comprehending the pathophysiology of genetic diseases and creating innovative treatment approaches customized for specific patients. This review aimed to present a thorough analysis of the current advancements in organoid technology and its biomedical applications for genetic diseases. We examined techniques for modeling genetic disorders using organoid platforms, analyze the approaches for incorporating genetic disease organoids into clinical practice, and showcase current breakthroughs in preclinical application, individualized healthcare, and transplantation. Through the integration of knowledge from several disciplines, such as genetics, regenerative medicine, and biological engineering, our aim is to enhance our comprehension of the complex connection between genetic variations and organoid models in relation to human health and disease.

Urine-derived stem cells (USCs) are adult human stem cells that can be collected noninvasively from urine and cultured in vitro. Because of their renal origin and reported therapeutic effects, we hypothesized that USCs would home to the injured kidney in acute kidney injury (AKI) models. We used mouse models of glycerol-induced rhabdomyolysis or unilateral nephrectomy with clamping ischemia reperfusion injury to model AKI. To track USC homing by live animal imaging, we administered luciferase-expressing (Luc) USCs to mice by intraperitoneal injection. We observed USC localization to both the tubules and glomeruli of injured mice within 3 h by histology. We confirmed the presence of Luc-USCs in the kidney at 3 h, 24 h, and 48 h after the injection using biodistribution analysis of quantitative bioluminescence tomography imaging. We performed immunostaining for kidney injury molecule-1 (KIM-1/HAVCR1/TIM-1) for kidney injury and found reduced expression in USC-treated group at 24 h after injection. To evaluate the effects of the human USCs on injured human nephrons, we injured human kidney organoids with the nephrotoxin cisplatin (5 μM) followed by 5 × 104 USC treatment. USCs were incorporated and lowered expression of KIM-1 in the organoids. USCs home to injured nephrons and reduce measures of kidney injury.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common renal genetic disease, with most patients carrying mutations in PKD1. The main feature is the formation of bilateral renal cysts, leading to end stage renal failure in a significant proportion of those affected. Despite recent advances made in understanding ADPKD, there are currently no effective curative therapies. The emergence of human induced pluripotent stem cell (hiPSC)-derived kidney disease models has led to renewed hope that more physiological systems will allow for the development of novel treatments. hiPSC-derived organoid models have been used to recapitulate ADPKD, however they present numerous limitations which remain to be addressed. In the present study, we report an efficient method for generating organoids containing a network of polarised and ciliated epithelial tubules. PKD1 null (PKD1-/-) organoids spontaneously develop dilated tubules, recapitulating early ADPKD cystogenesis. Furthermore, PKD1-/- tubules present primary cilia defects when dilated. Our model could therefore serve as a valuable tool to study early ADPKD cystogenesis and to develop novel therapies.

The rapidly developing field of renal spheroids and organoids has emerged as a valuable tool for modeling nephrotoxicity, kidney disorders, and kidney development. However, existing studies have relied on intricate and sophisticated differentiation protocols to generate organoids and tubuloids, necessitating the external administration of multiple growth factors within precise timeframes. In our study, we demonstrated that human adult renal progenitor cells (ARPCs) isolated from the urine of both healthy subjects and patients can form spheroids that naturally generated very long tubule-like structures. Importantly, the generation of these tubule-like structures is driven solely by ARPCs, without the need for the external use of chemokines or growth factors to artificially induce this process. These tubule-like structures exhibit the expression of structural and functional renal tubule markers and bear, in some cases, striking structural similarities to various nephron regions, including the distal convoluted tubule, the loop of Henle, and proximal convoluted tubules. Furthermore, ARPC spheroids express markers typical of pluripotent cells, such as stage-specific embryonic antigen 4 (SSEA4), secrete elevated levels of renin, and exhibit angiogenic properties. Notably, ARPCs isolated from the urine of patients with IgA nephropathy form spheroids capable of recapitulating the characteristic IgA1 deposition observed in this disease. These findings represent significant advancements in the field, opening up new avenues for regenerative medicine in the study of kidney development, mechanisms underlying renal disorders, and the development of regenerative therapies for kidney-related ailments.

Gene editing technology involves modifying target genes to alter genetic traits and generate new phenotypes. Beginning with zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), the field has evolved through the advent of clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR-Cas) systems, and more recently to base editors (BE) and prime editors (PE). These innovations have provided deep insights into the molecular mechanisms of complex biological processes and have paved the way for novel therapeutic strategies for a range of diseases. Gene editing is now being applied in the treatment of both genetic and acquired kidney diseases, as well as in kidney transplantation and the correction of genetic mutations. This review explores the current applications of mainstream gene editing technologies in biology, with a particular emphasis on their roles in kidney disease research and treatment of. It also addresses the limitations and challenges associated with these technologies, while offering perspectives on their future potential in this field.

The kidneys are vital for maintaining bodily homeostasis and are susceptible to various diseases that disrupt their function. Traditionally, research on kidney diseases has relied on animal models and simplistic two-dimensional cell cultures, which do not fully replicate human tissue pathology. To address this, recent advances focus on developing advanced 3D biomimeticin vitromodels using human-derived cells. These models mimic healthy and diseased kidney tissues with specificity, replicating key elements like glomerular and tubular structures through tissue engineering. By closely mimicking human physiology, they provide a promising platform for studying renal disorders, drug-induced nephrotoxicity, and evaluating new therapies. However, the challenges include optimizing scalability, reproducibility, and long-term stability to enhance reliability in research and clinical applications. This review highlights the transformative potential of 3D biomimeticin vitrokidney models in advancing biomedical research and clinical applications. By focusing on human-specific cell cultures and tissue engineering techniques, these models aim to overcome the limitations of conventional animal models and simplistic 2D cell cultures. The review discusses in detail the various types of biomimetic kidney models currently under development, their specific applications, and the innovative approaches used to construct them. It also addresses the challenges and limitations associated with these models for their widespread adoption and reliability in research settings.

Organoids have revolutionized in vitro research by offering three-dimensional, multicellular systems that recapitulate the structure, function, and genetics of human tissues. Initially developed from both pluripotent stem cells (PSCs) and adult stem cells (AdSCs), organoids have expanded to model nearly every major human organ, significantly advancing developmental biology, disease modeling, and therapeutic screening. This review highlights the progression of organoid technologies, emphasizing the integration of genetic tools, including CRISPR-Cas9, prime editing, and lineage tracing. These advancements have facilitated precise modeling of human-specific pathologies and drug responses, often surpassing traditional 2D cultures and animal models in accuracy. Emerging technologies, such as organoid fusion, xenografting, and optogenetics, are expected to further enhance our understanding of cellular interactions and microenvironmental dynamics. As organoid complexity and genetic engineering methods continue to evolve, they will become increasingly indispensable for personalized medicine and translational research, bridging gaps between in vitro and in vivo systems.

Kidney transplantation remains a pivotal treatment modality for kidney disease, yet its progress is significantly hindered by the scarcity of donor kidneys and ethical dilemmas surrounding their procurement. As organoid technology evolves and matures, the creation of bionic human kidney organoids offers profound potential for advancing kidney disease research, drug nephrotoxicity screening, and regenerative medicine. Nevertheless, current kidney organoid models grapple with limitations such as constrained cellular differentiation, underdeveloped functional structures, and a crucial absence of vascularization. This deficiency in vascularization, in particular, stunts organoid development, restricts their size, diminishes filtration capabilities, and may trigger immune inflammatory reactions through the resulting ischemic microenvironment. Hence, the achievement of vascularization within kidney organoids and the successful establishment of functional microvascular networks constitutes a paramount goal for their future progression. In this review, we provide an overview of recent advancements in biotechnology domains, encompassing organ-on-a-chip technology, biomimetic matrices, and bioprinting, with the aim of catalyzing technological breakthroughs that can enhance the vascularization of kidney organoids and broaden their applicability. These technologies hold the key to unlocking the full potential of kidney organoids as a transformative therapeutic option for kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a prevalent hereditary disorder characterized by distinct phenotypic variability that has posed challenges for advancing in-depth research. Recent advancements in kidney organoid construction technologies have enabled researchers to simulate kidney development and create simplified in vitro experimental environments, allowing for more direct observation of how genetic mutations drive pathological phenotypes and disrupt physiological functions. Emerging technologies, such as microfluidic bioreactor culture systems and single-cell transcriptomics, have further supported the development of complex ADPKD organoids, offering robust models for exploring disease mechanisms and facilitating drug discovery. Nevertheless, significant challenges remain in constructing more accurate ADPKD disease models. This review will summarize recent advances in ADPKD organoid construction, focusing on the limitations of the current techniques and the critical issues that need to be addressed for future breakthroughs. New and Noteworthy: This review presents recent advancements in ADPKD organoid construction, particularly iPSC-derived models, offering new insights into disease mechanisms and drug discovery. It focuses on challenges such as limited vascularization and maturity, proposing potential solutions through emerging technologies. The ongoing optimization of ADPKD organoid models is expected to enhance understanding of the disease and drive breakthroughs in disease mechanisms and targeted therapy development.

A drug to be successfully launched in the market requires a significant amount of capital, resources and time, where the unsuccessful results in the last stages lead to catastrophic failure for discovering drugs. This is the very reason which calls for the invention of innovative models that can closely mimic the human in vivo model for producing reliable results. Throughout the innovation line, there has been improvement in the rationale in silico designing but yet there is requirement for in vitro-in vivo correlations. During the evolving of the drug testing models, the 3D models produced by different methods have been proven to produce better results than the traditional 2D models. However, the in vitro fabrications of live tissues are still bottleneck in realizing their complete potential. There is an urgent need for the development of single, standard and simplified in vitro 3D tissue models that can be reliable for investigating the biological and pathological aspects of drug discovery, which is yet to be achieved. The existing pre-clinical models have considerable drawbacks despite being the gold standard in pre-clinical research. The major drawback being the interspecies differences and low reliability on the generated results. This gap could be overcome by the fabrication of bioengineered human disease models for drug screening. The advancement in the fabrication of 3D models will provide a valuable tool in screening drugs at different stages as they are one step closer to bio-mimic human tissues. In this review, we have discussed on the evolution of preclinical studies, and different models, including mini tissues, spheroids, organoids, bioengineered three dimensional models and organs on chips. Furthermore, we provide details of different disease models fabricated across various organs and their applications. In addition to this, the review also focuses on the limitations and the current prospects of the role of three dimensionally bioprinted models in drug screening and development.

The mammalian target of rapamycin (mTOR) pathway is a therapeutic target in polycystic kidney disease (PKD), but mTOR inhibitors such as everolimus have failed to show efficacy at tolerated doses in clinical trials. Here, we introduce AV457, a novel rapalog developed to reduce side effects, and assess its dose-dependent safety and efficacy versus everolimus in PKD1-/- and PKD2-/- human kidney organoids, which form cysts in a PKD-specific way. Both AV457 and everolimus reduce cyst growth over time. At intermediate doses, AV457 exhibits an improved safety profile relative to everolimus, with comparable efficacy. Target engagement assays confirm mTOR pathway inhibition and greater selectivity of AV457 for mTOR complex 1 versus complex 2, compared to everolimus. AV457 thus provides a more favorable balance of safety and efficacy for PKD compared to everolimus and merits further consideration as an investigational therapeutic.

The increasing incidence of kidney diseases has highlighted the need for in vitro experimental models to mimic disease development and to test new therapeutic approaches. Traditional two-dimensional in vitro experimental models are not fully able to recapitulate renal diseases. Instead, kidney organoids represent three-dimensional models that better mimic the human organ from both structural and functional points of view. Human pluripotent stem cells (PSCs), both embryonic and induced, are ideal sources for generating renal organoids. These organoids contain all renal cell types and the protocols to differentiate PSCs into renal organoids consist of three different stages that recapitulate embryonic development: mesodermal induction, nephron progenitor formation, and nephron differentiation. Recently it has been establish a renal organoid model where collecting ducts are also present. In this case, the presence of ureteric bud progenitor cells is essential. Renal organoids are particularly useful for studying genetic diseases, by introducing the specific mutations in PSCs by genome editing or generating organoids from patient-derived PSCs. Moreover, renal organoids represent promising models in toxicology studies and testing new therapeutic approaches. Renal organoids can be established also from adult stem cells. This type of organoid, named tubuloid, is composed only of epithelial cells and recapitulates the tissue repair process. The tubuloids can be generated from adult stem or progenitor cells, obtained from renal biopsies or urine, and are promising in vitro models for studying tubular functions, diseases, and regeneration. Tubuloids can be derived from patients and permit the study of genetic diseases, performing personalized drug screening and modeling renal pathologies.

Human pluripotent stem cell-derived kidney organoids hold promise for future applications in regenerative medicine. However, significant biological hurdles need to be overcome to enable their use as a transplantable stem cell-derived therapeutic graft. Current kidney organoid protocols do not recapitulate a complete integrated developing kidney, but embryonic kidney transplantations have provided clues for advancing maturation and functionality of kidney organoids. Transplantation, subsequent vascularization, and blood perfusion of kidney organoids improve nephron patterning and maturation, suggesting a role for angiocrine factors as well as metabolic wiring in these processes. Transplanted organoids exhibit filtration, but the resulting filtrate has no apparent exit path for excretion. Improved in vitro patterning of kidney organoids may be required such that a more structurally correct tissue is formed before transplant. Here we review current progress with transplantation of kidney organoids, as well as their engraftment and integration, and identify the key obstacles to therapeutic success and how these might be achieved.

Organoids are 3D in vitro models that fulfill a hierarchical function, representing a small version of living tissues and, therefore, a good approximation of cellular mechanisms. However, one of the main disadvantages of these models is the appearance of a necrotic core due to poor vascularization. The aim of this work is the development of a numerical framework that incorporates the mechanical stimulation as a key factor in organoid vascularization. Parameters, such as fluid velocity and nutrient consumption, are analyzed along the organoid evolution.

The mathematical model created for this purpose combines continuum and discrete approaches. In the continuum part, the fluid flow and the diffusion of oxygen and nutrients are modeled using a finite element method approach. Meanwhile, the growth of the organoid, blood vessel evolution, as well as their interaction with the surrounding environment, are modeled using agent-based methods.

Continuum model outcomes include the distribution of shear stress, pressure and fluid velocity around the organoid surface, in addition to the concentration of oxygen and nutrients in its interior. The agent models account for cell proliferation, differentiation, organoid growth and blood vessel morphology, for the different case studies considered.

Two main conclusions are achieved in this work: (i) the results of the study quantitatively predict in vitro data, with an enhanced blood vessel invasion under high fluid flow and (ii) the diffusion and consumption model parameters of the organoid cells determine the thickness of the proliferative, quiescent, hypoxic and necrotic layers.

Nephron formation and patterning are driven by complex cell biology. Progenitors migrate, transition into epithelia, and generate an axial epithelial polarity with distinct transcriptional signatures, regulating virtually all physiologies of the maturing kidney post birth. Here we review current insights into mammalian nephrogenesis and discuss how the nephron forms and patterns along its proximal-distal axis during embryonic and fetal development. Genetic pathways that are necessary for this process are discussed and integrated into the cell biology and morphogenetic programs underpinning nephrogenesis. Together, these views outline a developmental blueprint for replicating nephron formation in vitro.

Renal progenitor organoids have been proposed as a source of tissue for kidney regeneration; however, their clinical translatability has not been demonstrated due to an inability to mass-produce comprehensive renal progenitor organoids and the lack of an effective intra-renal delivery platform that facilitates rapid integration into functionally meaningful sites. This study addresses these shortcomings. Human-induced pluripotent stem cells were differentiated into renal progenitor cells using an established protocol and aggregated using a novel assembly method to produce high yields of organoids. Organoids were encapsulated in collagen-based scaffolds for in vitro study and in vivo implantation into mouse renal cortex. In vitro, the organoids demonstrated sustained cell viability and renal structure maturation over time. In vivo delivered organoids showed rapid integration into host renal parenchyma while showing tubular and glomerular-like structure development and maturity markers. This proof-of-concept study presents many promising results, providing a system of renal organoid formation and delivery that may support the development of clinically translatable therapies and the advancement of in vitro renal organoid studies.

Organoids are miniature organs developed through technology. Kidney organoids that originate from human inducible pluripotent stem cells (iPSCs) were developed to recreate renal diseases. However, it is impossible to simultaneously produce kidney organoids from iPSCs of multiple individuals and in a single medium. We herein report the development of adult renal tubular organoids, namely, "tubuloids," from primary renal epithelial cells from multiple human individuals in a single medium.

Kidneys from eight patients who underwent nephrectomy due to malignancy were sectioned, and primary renal epithelial tubule cells were cultured; four had normal kidney function, and four had mild chronic kidney disease (CKD). Growth factors and Matrigel were added to the primary culture.

Primary cultured renal epithelial cells from normal kidneys exhibited a fine and swollen epithelial appearance, whereas those from kidneys with mild CKD were smaller and slightly elongated. Growth was faster in normal kidney cells than in mild CKD cells. At the beginning of the three-dimensionalization (day 0), normal renal tubuloids grew faster than mild CKD tubuloids. The difference in size between normal and mild CKD tubuloids was not obvious by day 5. Both tubuloid types had comparable sizes by day 21.

Renal tubular organoids can be developed simultaneously and in a single medium. Our method is expected to be used as a human pathological model.

Precision-cut kidney slices (PCKS) provide a powerful model to close the gap between in vivo and in vitro research. Publications by various authors favor different incubation conditions, media, and antibiotics, that have not yet been compared in a standardized manner. After preparation, rat-PCKS were incubated in a total of nine combinations of incubation media and antibiotics for four days. We found that a combination of DMEM/F-12 and gentamicin showed the highest levels of viability. Utilizing both qualitative and quantitative methods, we observed stable levels of cellular viability for 10 days when incubated in the most suitable medium combination of DMEM and gentamicin. Additionally, a calcein acetoxymethyl/ethidium homodimer-1 based live/dead staining, analysis of total protein content and lactate dehydrogenase (LDH) were explored to assess both short- and long-term tissue viability. PCKS showed a significant decrease in total protein content, leveling off at around 60% over the duration of 10 days. To be able to evaluate viability irrespective of decreases in total protein detected, we chose to utilize the alamarBlue Cell Viability Assay. Quantifying both intra- and extracellular activity of LDH, while using different concentrations of ethanol as a positive control, we explored enzyme content as a parameter for cell membrane damage and cytotoxicity in PCKS. Overall, we showed that PCKS are suitable for both short- and long-term observation by optimizing incubation parameters, with numerous possibilities for other assays and methods in future studies.

To date, genetically modified organoids are emerging as a promising 3D modeling tool aimed at solving genetically relevant clinical and biomedical problems for regenerative medicine and tissue engineering. As an optimal vehicle for gene delivery, genetically modified organoids can enhance or reduce the expression of target genes through virus and non-virus-based gene transfection methods to achieve tissue regeneration. Animal experiments and preclinical studies have demonstrated the beneficial role of genetically modified organoids in various aspects of organ regeneration, including thymus, lacrimal glands, brain, lung, kidney, photoreceptors, etc. Furthermore, the technology offers a potential treatment option for various diseases, such as Fabry disease, non-alcoholic steatohepatitis, and Lynch syndrome. Nevertheless, the uncertain safety of genetic modification, the risk of organoid application, and bionics of current genetically modified organoids are still challenging. This review summarizes the researches on genetically modified organoids in recent years, and describes the transfection methods and functions of genetically modified organoids, then introduced their applications at length. Also, the limitations and future development directions of genetically modified organoids are included.

Kidney disease has become a growing public health problem worldwide, and there is an urgent need to develop reliable models for investigating novel and effective treatment strategies. In recent years, kidney organoids, as novel models different from traditional two-dimensional cells and model animals, have attracted more and more attention. Current advances have allowed the generation of kidney organoids from the directed differentiation of induced pluripotent stem cells (iPSCs), which possess similar characteristics to embryonic stem cells, but bypass ethical constraints and have a wide range of sources.

Herein, the methods of generating renal organoids from iPSCs, the applications of iPSC-derived renal organoids in disease modeling, drug effectiveness detection, and regenerative medicine as well as the challenges were reviewed.

iPSC-derived renal organoids can be used to model kidney diseases and are great models for studying kidney injury and toxicity. Many efforts are needed to finally apply organoids into clinical application.

Kidney organoids are connected to the host circulation and mature after transplantation. However, they are still immature compared to the adult kidneys, and their precise maturation stages remain unclear. By transplanting the mouse embryonic kidney as a model system for organoid transplantation, we report here the maturation defects of the graft, especially in the medulla. Single cell profiling of the developing kidneys in vivo identified gene sets associated with the maturation of the collecting duct epithelium and medullary stroma. These data revealed an upregulation of genes associated with channel/transporter functions and immune defense, as well as a downregulation of neuronal genes. Using these marker genes, we found that the maturation of the collecting duct and medullary stroma in the grafts barely corresponds to the perinatal stage, which was confirmed histologically by using representative genes. Thus, the gene sets obtained serve as maturation coordinates for the renal medulla and will be helpful in analyzing its maturation defects after transplantation. They will also provide a useful basis for further maturation of transplanted kidney organoids.

Kidney cancer is a significant global health problem that affects nearly 1 in 25 of cancer patients. Prevalence, morbidity and mortality data associated with kidney cancer continue to increase every year, revealing a heavy economic and social burden. Organoid culture is a new research tool with great potential for many applications, particularly in cancer research. The integration of organoids with other emerging technologies has simultaneously expanded their potential applications. However, there is no thorough assessment of organoids in the field of renal cancer research.

This paper presents a comprehensive review of the current development of renal cancer organoids and discusses the corresponding solutions and future directions of renal cancer organoids.

In this study, we have compared the operating procedures of different organoid culture protocols and proposed a summary of constituents in culture media. Extensive discussions of renal cancer organoids, including generation and maintenance approaches, application scenarios, current challenges and prospects, have also been made. The information required for this study is extracted from literature databases such as PubMed, SCOPUS and Web of Science.

In this article, we systematically review thirteen successful methods for generating organoids to kidney cancer and provide practical guidelines for their construction as a reference. In addition, we also elucidate the clinical application of organoids, address the existing challenges and limitations, and highlight promising prospects.

Ultimately, we firmly believe that as kidney tumour organoids continue to develop and improve, they will become a crucial tool for treating kidney cancer.

Cilia are membrane-bound organelles found on the surface of most mammalian cell types and play numerous roles in human physiology and development, including osmo- and mechanosensation, as well as signal transduction. Ciliopathies are a large group of, usually rare, genetic disorders resulting from abnormal ciliary structure or ciliary dysfunction that have a high collective prevalence. Autosomal dominant or recessive polycystic kidney disease (ADPKD/ARPKD), Bardet-Biedl-Syndrome, and primary ciliary dyskinesia (PCD) are the most frequent etiologies. Rodent and zebrafish models have improved the understanding of ciliopathy pathophysiology. Yet, the limitations of these genetically modified animal strains include the inability to fully replicate the phenotypic heterogeneity found in humans, including variable multiorgan involvement. Organoids, self-assembled three-dimensional cell-based models derived from human induced pluripotent stem cells (iPSCs) or primary tissues, can recapitulate certain aspects of the development, architecture, and function of the target organ "in the dish." The potential of organoids to model patient-specific genotype-phenotype correlations has increased their popularity in ciliopathy research and led to the first preclinical organoid-based ciliopathy drug screens. This review comprehensively summarizes and evaluates current ciliopathy organoid models, focusing on kidney, airway, liver, and retinal organoids, as well as the specific methodologies used for their cultivation and for interrogating ciliary dysfunction.

Cisplatin is a common platinum-based chemotherapeutic that induces acute kidney injury (AKI) in about 30% of patients. Pharmacokinetic/toxicodynamic (PKTD) models of cisplatin-induced AKI have been used to understand risk factors and evaluate potential mitigation strategies. While both traditional clinical biomarkers of kidney function [e.g., serum creatinine (SCr), blood urea nitrogen (BUN), estimated glomerular filtration rate (eGFR), and creatinine clearance (CrCl)] and newer subclinical biomarkers of kidney injury [e.g., urinary kidney injury molecule 1 (KIM-1), beta-2 microglobulin (B2M), neutrophil gelatinase-associated lipocalin (NGAL), calbindin, etc.] can be used to detect cisplatin-induced AKI, published PKTD models are limited to using only traditional clinical biomarkers. Previously identified risk factors for cisplatin nephrotoxicity have included dose, age, sex, race, body surface area, genetics, concomitant medications, and comorbid conditions. However, the relationships between concentrations and the pharmacokinetics (PK) of platinum and biomarkers of kidney injury have not been well elucidated. This review discusses the evaluation of cisplatin-induced nephrotoxicity in clinical studies, mouse models, and in vitro models, and examines the available human PK and toxicodynamic (TD) data. Improved understanding of the relationships between platinum PK and TD, in the presence of identified risk factors, will enable the prediction and prevention of cisplatin kidney injury. SIGNIFICANCE STATEMENT: As cisplatin treatment continues to cause AKI in a third of patients, it is critical to improve the understanding of the relationships between platinum PK and nephrotoxicity as assessed by traditional clinical and contemporary subclinical TD markers of kidney injury. Prediction and prevention of cisplatin-induced nephrotoxicity will be advanced by the evolving development of PKTD models that incorporate kidney injury biomarkers with enhanced sensitivity and include covariates that can impact risk of developing cisplatin-induced AKI.

Organoids are "mini-organs" that self-organize and differentiate from stem cells under in vitro 3D culture conditions, mimicking the spatial structure and function of tissues in vivo. Extracellular vesicles (EVs) are nanoscale phospholipid bilayer vesicles secreted by living cells, rich in bioactive molecules, with excellent biocompatibility and low immunogenicity. Compared to EVs, organoid-derived EVs (OEVs) exhibit higher yield and enhanced biological functions. Organoids possess stem cell characteristics, and OEVs are capable of delivering active substances, making both highly promising for medical applications. In this review, we provide an overview of the fundamental biological principles of organoids and OEVs, and discuss their current applications in disease treatment. We then focus on the differences between OEVs and traditional EVs. Subsequently, we present methods for the engineering modification of OEVs. Finally, we critically summarize the advantages and challenges of organoids and OEVs. In conclusion, we believe that a deeper understanding of organoids and OEVs will provide innovative solutions to complex diseases.

The kidney plays a crucial role in maintaining the body's microenvironment homeostasis. However, current treatment options and therapeutic agents for chronic kidney disease (CKD) are limited. Fortunately, the advent of kidney organoids has introduced a novel in vitro model for studying kidney diseases and drug screening. Despite significant efforts has been leveraged to mimic the spatial-temporal dynamics of fetal renal development in various types of kidney organoids, there is still a discrepancy in cell types and maturity compared to native kidney tissue. The extracellular matrix (ECM) plays a crucial role in regulating cellular signaling, which ultimately affects cell fate decision. As a result, ECM can refine the microenvironment of organoids, promoting their efficient differentiation and maturation. This review examines the existing techniques for culturing kidney organoids, evaluates the strengths and weaknesses of various types of kidney organoids, and assesses the advancements and limitations associated with the utilization of the ECM in kidney organoid culture. Additionally, it presents a discussion on constructing specific physiological and pathological microenvironments using decellularized extracellular matrix during certain developmental stages or disease occurrences, aiding the development of kidney organoids and disease models.

Progression of cystic kidney disease has been linked to activation of the mTORC1 signaling pathway. Yet the utility of mTORC1 inhibitors to treat patients with polycystic kidney disease remains controversial despite promising preclinical data. To define the cell intrinsic role of mTORC1 for cyst development, the mTORC1 subunit gene Raptor was selectively inactivated in kidney tubular cells lacking cilia due to simultaneous deletion of the kinesin family member gene Kif3A. In contrast to a rapid onset of cyst formation and kidney failure in mice with defective ciliogenesis, both kidney function, cyst formation discerned by magnetic resonance imaging and overall survival were strikingly improved in mice additionally lacking Raptor. However, these mice eventually succumbed to cystic kidney disease despite mTORC1 inactivation. In-depth transcriptome analysis revealed the rapid activation of other growth-promoting signaling pathways, overriding the effects of mTORC1 deletion and identified cyclin-dependent kinase (CDK) 4 as an alternate driver of cyst growth. Additional inhibition of CDK4-dependent signaling by the CDK4/6 inhibitor Palbociclib markedly slowed disease progression in mice and human organoid models of polycystic kidney disease and potentiated the effects of mTORC1 deletion/inhibition. Our findings indicate that cystic kidneys rapidly adopt bypass mechanisms typically observed in drug resistant cancers. Thus, future clinical trials need to consider combinatorial or sequential therapies to improve therapeutic efficacy in patients with cystic kidney disease.

The application of organoids has been limited by the lack of methods for producing uniformly mature organoids at scale. This study introduces an organoid culture platform, called UniMat, which addresses the challenges of uniformity and maturity simultaneously. UniMat is designed to not only ensure consistent organoid growth but also facilitate an unrestricted supply of soluble factors by a 3D geometrically-engineered, permeable membrane-based platform. Using UniMat, we demonstrate the scalable generation of kidney organoids with enhanced uniformity in both structure and function compared to conventional methods. Notably, kidney organoids within UniMat show improved maturation, showing increased expression of nephron transcripts, more in vivo-like cell-type balance, enhanced vascularization, and better long-term stability. Moreover, UniMat's design offers a more standardized organoid model for disease modeling and drug testing, as demonstrated by polycystic-kidney disease and acute kidney injury modeling. In essence, UniMat presents a valuable platform for organoid technology, with potential applications in organ development, disease modeling, and drug screening.

Kidney organoids, replicating human development, pathology, and drug responses, are a promising model for advancing bioscience and pharmaceutical innovation. However, reproducibility, accuracy, and quantification challenges hinder their broader utility for advanced biological and pharmaceutical applications. Herein, we present a dynamic kidney organoid microphysiological analysis platform (MAP), designed to enhance organoid modeling and assays within physiologically relevant environments, thereby expanding their utility in advancing kidney physiology and pathology research. First, precise control of the dynamic microenvironment in MAP enhances the ability to fine-tune nephrogenic intricacies, facilitating high-throughput and reproducible human kidney organoid development. Also, MAP's miniaturization of kidney organoids significantly advances pharmaceutical research by allowing for detailed analysis of entire nephron segments, which is crucial for assessing the nephrotoxicity and safety of drugs. Furthermore, the MAP's application in disease modeling faithfully recapitulates pathological development and functions as a valuable testbed for therapeutic exploration in polycystic kidney diseases. We envision the kidney organoid MAP enhancing pharmaceutical research, standardizing processes, and improving analytics, thereby elevating the quality and utility of organoids in biology, pharmacology, precision medicine, and education.

Organophosphate (OP) poisoning, both accidental and with suicidal intent, is a global medical challenge. While the primary toxicity of these pesticides is based on the inhibition of acetylcholinesterase (AChE), case reports describe patients developing OP-mediated renal insufficiency. We set out to investigate possible pathomechanisms utilizing rat precision-cut kidney slices (PCKS). Depending on the method of investigation, PCKS were observed for a maximum of 10 days. PCKS exposed to OP compounds (malaoxon, malathion, paraoxon, parathion) showed a dose-dependent loss of viability and a reduction of total protein content over the course of 10 days. A concentration of 500 µM OP showed the most differences between OP compounds. After two days of incubation parathion showed a significantly lower level of viability than malathion. The respective effects of paraoxon and malaoxon were not significantly different from the control. However, effects of OP were only observed in concentrations exceeding those that were needed to achieve significant AChE inhibition in rat kidney tissue. In addition, we observed histological changes, without inducing LDH leakage. Overall, results suggest that OP exert effects in kidney tissue, that exceed those expected from the sole inhibition of AChE and vary between compounds. Without signs of necrosis, findings call for studies that address other possible pathomechanisms, including inflammatory response, oxidative stress or activation of apoptosis to further understand the nephrotoxicity of OP compounds. Monitoring oxon concentration over time, we demonstrated reduced enzyme-inhibiting properties in the presence of PCKS, suggesting interactions between OP compound and kidney tissue.

Acute kidney injury (AKI) and chronic kidney disease (CKD) are significant public health issues associated with a long-term increase in mortality risk, resulting from various etiologies including renal ischemia, sepsis, drug toxicity, and diabetes mellitus. Numerous preclinical models have been developed to deepen our understanding of the pathophysiological mechanisms and therapeutic approaches for kidney diseases. Among these, rodent models have proven to be powerful tools in the discovery of novel therapeutics, while the development of kidney organoids has emerged as a promising advancement in the field. This review provides a comprehensive analysis of the construction methodologies, underlying biological mechanisms, and recent therapeutic developments across different AKI and CKD models. Additionally, this review summarizes the advantages, limitations, and challenges inherent in these preclinical models, thereby contributing robust evidence to support the development of effective therapeutic strategies.

The three-dimensional (3D) kidney organoid is a breakthrough model for recapitulating renal morphology and function in vitro, which is grown from stem cells and resembles mammalian kidney organogenesis. Currently, protocols for cultivating this model from induced pluripotent stem cells (iPSCs) and patient-derived adult stem cells (ASCs) have been widely reported. In recent years, scientists have focused on combining cutting-edge bioengineering and bioinformatics technologies to improve the developmental accuracy of kidney organoids and achieve high-throughput experimentation. As a remarkable tool for mechanistic research of the renal system, kidney organoid has both potential and challenges. In this review, we have described the evolution of kidney organoid establishment methods and highlighted the latest progress leading to a more sophisticated kidney transformation research model. Finally, we have summarized the main applications of renal organoids in exploring kidney disease.

Kidney organoids - 3D representations of kidneys made either from pluripotent or tissue stem cells - have been available for well over a decade. Their application could confer notable benefits over longstanding in vivo approaches with the potential for clinically aligned human cells and reduced ethical burdens. They been used, at a proof-of-concept level, in development in disease modeling (including with patient-derived stem cells), and in screening drugs for efficacy/toxicity. They differ from real kidneys: they represent only foetal-stage tissue, in their simplest forms they lack organ-scale anatomical organization, they lack a properly arranged vascular system, and include non-renal cells. Cell specificity may be improved by better techniques for differentiation and/or sorting. Sequential assembly techniques that mimic the sequence of natural development, and localized sources of differentiation-inducing signals, improve organ-scale anatomy. Organotypic vascularization remains a challenge: capillaries are easy, but the large vessels that should serve them are absent from organoids and, even in cultured real kidneys, these large vessels do not survive without blood flow. Transplantation of organoids into hosts results in their being vascularized (though probably not organotypically) and in some renal function. It will be important to transplant more advanced organoids, with a urine exit, in the near future to assess function more stringently. Transplantation of human foetal kidneys, followed by nephrectomy of host kidneys, keeps rats alive for many weeks, raising hope that, if organoids can be produced even to the limited size and complexity of foetal kidneys, they may one day be useful in renal replacement.

Recent years have seen the creation and popularization of various complexin vitromodels (CIVMs), such as organoids and organs-on-chip, as a technology with the potential to reduce animal usage in pharma while also enhancing our ability to create safe and efficacious drugs for patients. Public awareness of CIVMs has increased, in part, due to the recent passage of the FDA Modernization Act 2.0. This visibility is expected to spur deeper investment in and adoption of such models. Thus, end-users and model developers alike require a framework to both understand the readiness of current models to enter the drug development process, and to assess upcoming models for the same. This review presents such a framework for model selection based on comparative -omics data (which we term model-omics), and metrics for qualification of specific test assays that a model may support that we term context-of-use (COU) assays. We surveyed existing healthy tissue models and assays for ten drug development-critical organs of the body, and provide evaluations of readiness and suggestions for improving model-omics and COU assays for each. In whole, this review comes from a pharma perspective, and seeks to provide an evaluation of where CIVMs are poised for maximum impact in the drug development process, and a roadmap for realizing that potential.

The KCNJ16 gene has been associated with a novel kidney tubulopathy phenotype, viz. disturbed acid-base homeostasis, hypokalemia and altered renal salt transport. KCNJ16 encodes for Kir5.1, which together with Kir4.1 constitutes a potassium channel located at kidney tubular cell basolateral membranes. Preclinical studies provided mechanistic links between Kir5.1 and tubulopathy, however, the disease pathology remains poorly understood. Here, we aimed at generating and characterizing a novel advanced in vitro human kidney model that recapitulates the disease phenotype to investigate further the pathophysiological mechanisms underlying the tubulopathy and potential therapeutic interventions.

We used CRISPR/Cas9 to generate KCNJ16 mutant (KCNJ16+/- and KCNJ16-/-) cell lines from healthy human induced pluripotent stem cells (iPSC) KCNJ16 control (KCNJ16WT). The iPSCs were differentiated following an optimized protocol into kidney organoids in an air-liquid interface.

KCNJ16-depleted kidney organoids showed transcriptomic and potential functional impairment of key voltage-dependent electrolyte and water-balance transporters. We observed cysts formation, lipid droplet accumulation and fibrosis upon Kir5.1 function loss. Furthermore, a large scale, glutamine tracer flux metabolomics analysis demonstrated that KCNJ16-/- organoids display TCA cycle and lipid metabolism impairments. Drug screening revealed that treatment with statins, particularly the combination of simvastatin and C75, prevented lipid droplet accumulation and collagen-I deposition in KCNJ16-/- kidney organoids.

Mature kidney organoids represent a relevant in vitro model for investigating the function of Kir5.1. We discovered novel molecular targets for this genetic tubulopathy and identified statins as a potential therapeutic strategy for KCNJ16 defects in the kidney.

Recent advances in CRISPR-Cas systems have revolutionised the study and treatment of kidney diseases, including acute kidney injury (AKI), chronic kidney disease (CKD), diabetic kidney disease (DKD), lupus nephritis (LN), and polycystic kidney disease (PKD). CRISPR-Cas technology offers precise and versatile tools for genetic modification in monogenic kidney disorders such as PKD and Alport syndrome. Recent advances in CRISPR technology have also shown promise in addressing other kidney diseases like AKI, CKD, and DKD. CRISPR-Cas holds promise to edit genetic mutations underlying these conditions, potentially leading to more effective and long-lasting treatments. Furthermore, the adaptability of CRISPR-Cas systems allows for developing tailored therapeutic strategies that specifically target the genetic and molecular mechanisms contributing to different kidney diseases. Beyond DNA modifications, CRISPR-Cas technologies also enable editing noncoding RNA, such as lncRNAs and miRNAs, in kidney diseases. Despite these advancements, significant challenges persist, including delivery efficiency to specific kidney cells and potential off-target effects. However, the rapid progress in CRISPR-Cas technology suggests a transformative impact on the future management of kidney diseases, offering the potential for enhanced patient outcomes through personalised and precise therapeutic approaches. This chapter highlights the recent advancement of CRISPR-Cas systems and their potential applications in various kidney diseases.

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 liver is the most important metabolic organ in the body. While mouse models and cell lines have further deepened our understanding of liver biology and related diseases, they are flawed in replicating key aspects of human liver tissue, particularly its complex structure and metabolic functions. The organoid model represents a major breakthrough in cell biology that revolutionized biomedical research. Organoids are in vitro three-dimensional (3D) physiological structures that recapitulate the morphological and functional characteristics of tissues in vivo, and have significant advantages over traditional cell culture methods. In this review, we discuss the generation strategies and current advances in the field focusing on their application in regenerative medicine, drug discovery and modeling diseases.

Urological malignancy (UM) is among the leading threats to health care worldwide. Recent years have seen much investment in fundamental UM research, including mechanistic investigation, early diagnosis, immunotherapy, and nanomedicine. However, the results are not fully satisfactory. Bioprinted research models (BRMs) with programmed spatial structures and functions can serve as powerful research tools and are likely to disrupt traditional UM research paradigms. Herein, a comprehensive review of BRMs of UM is presented. It begins with a brief introduction and comparison of existing UM research models, emphasizing the advantages of BRMs, such as modeling real tissues and organs. Six kinds of mainstream bioprinting techniques used to fabricate such BRMs are summarized with examples. Thereafter, research advances in the applications of UM BRMs, such as culturing tumor spheroids and organoids, modeling cancer metastasis, mimicking the tumor microenvironment, constructing organ chips for drug screening, and isolating circulating tumor cells, are comprehensively discussed. At the end of this review, current challenges and future development directions of BRMs and UM are highlighted from the perspective of interdisciplinary science.