In Vivo Assessment of Laboratory-Grown Kidney Tissue Grafts.

Directed differentiation of stem cells is an attractive approach to generate kidney tissue for regenerative therapies. Currently, the most informative platform to test the regenerative potential of this tissue is engraftment into kidneys of immunocompromised rodents. Stem cell-derived kidney tissue is vascularized following engraftment, but the connection between epithelial tubules that is critical for urine to pass from the graft to the host collecting system has not yet been demonstrated. We show that one significant obstacle to tubule fusion is the accumulation of fibrillar collagens at the interface between the graft and the host. As a screening strategy to identify factors that can prevent this collagen accumulation, we propose encapsulating laboratory-grown kidney tissue in fibrin hydrogels supplemented with candidate compounds such as recombinant proteins, small molecules, feeder cells, and gene therapy vectors to condition the local graft environment. We demonstrate that the AAV-DJ serotype is an efficient gene therapy vector for the subcapsular region and that it is specific for interstitial cells in this compartment. In addition to the histological evaluation of epithelial tubule fusion, we demonstrate the specificity of two urine biomarker assays that can be used to detect human-specific markers of the proximal nephron (CD59) and the distal nephron (uromodulin), and we demonstrate the deposition of human graft-derived urine into the mouse collecting system. Using the testing platform described in this report, it will be possible to systematically screen factors for their potential to promote epithelial fusion of graft and host tissue with a functional intravital read-out.

The Extracellular Matrix Environment of Clear Cell Renal Cell Carcinoma.

The extracellular matrix (ECM) of tumors is a complex mix of components characteristic of the tissue of origin. In the majority of clear cell renal cell carcinomas (ccRCCs), the tumor suppressor VHL is inactivated. VHL controls matrix organization and its loss promotes a loosely organized and angiogenic matrix, predicted to be an early step in tumor formation. During tumor evolution, cancer-associated fibroblasts (CAFs) accumulate, and they are predicted to produce abundant ECM. The ccRCC ECM composition qualitatively resembles that of the healthy kidney cortex in which the tumor arises, but there are important differences. One is the quantitative difference between a healthy cortex ECM and a tumor ECM; a tumor ECM contains a higher proportion of interstitial matrix components and a lower proportion of basement membrane components. Another is the breakdown of tissue compartments in the tumor with mixing of ECM components that are physically separated in healthy kidney cortex. Numerous studies reviewed in this work reveal effects of specific ECM components on the growth and invasive behaviors of ccRCCs, and extrapolation from other work suggests an important role for ECM in controlling ccRCC tumor rigidity, which is predicted to be a key determinant of invasive behavior.

Targets for Renal Carcinoma Growth Control Identified by Screening FOXD1 Cell Proliferation Pathways.

Clinical association studies suggest that FOXD1 is a determinant of patient outcome in clear cell renal cell carcinoma (ccRCC), and laboratory investigations have defined a role for this transcription factor in controlling the growth of tumors through regulation of the G2/M cell cycle transition. We hypothesized that the identification of pathways downstream of FOXD1 may define candidates for pharmacological modulation to suppress the G2/M transition in ccRCC. We developed an analysis pipeline that utilizes RNA sequencing, transcription factor binding site analysis, and phenotype validation to identify candidate effectors downstream from FOXD1. Compounds that modulate candidate pathways were tested for their ability to cause growth delay at G2/M. Three targets were identified: FOXM1, PME1, and TMEM167A, which were targeted by compounds FDI-6, AMZ-30, and silibinin, respectively. A 3D ccRCC tumor replica model was used to investigate the effects of these compounds on the growth of primary cells from five patients. While silibinin reduced 3D growth in a subset of tumor replicas, FDI-6 reduced growth in all. This study identifies tractable pathways to target G2/M transition and inhibit ccRCC growth, demonstrates the applicability of these strategies across patient tumor replicas, and provides a platform for individualized patient testing of compounds that inhibit tumor growth.

Growth control of the kidney.

The functional mass of kidney tissue in an adult is an important determinant of human health. Kidney formation during development is an essential determinant of the final nephron endowment of the adult organ, and no evidence has been reported that mice or humans are able to generate new nephrons after the developmental period. Mechanisms controlling organ growth after development are essential to establish the final adult organ size. The potential for organ growth is maintained in adult life and the size of one kidney may be significantly increased by loss of the contralateral kidney. The mouse has provided a model system for investigators to critically explore genetic, cell biological, and hormonal control of developmental and juvenile kidney growth. This article reviews three basic aspects of kidney size regulation: (1) Mechanisms that control nephron formation and how these are altered by the cessation of nephrogenesis at the end of the developmental period. (2) Applicability of the general model for growth hormone-insulin like growth factor control to kidney growth both pre- and postnatally. (3) Commonalities between mechanisms of juvenile kidney growth and the compensatory growth that is stimulated in adult life by reduction of kidney mass. Understanding the mechanisms that determine set-points for cell numbers and size in the kidney may inform ongoing efforts to generate kidney tissue from stem cells.

Smad4 controls proliferation of interstitial cells in the neonatal kidney.

Expansion of interstitial cells in the adult kidney is a hallmark of chronic disease, whereas their proliferation during fetal development is necessary for organ formation. An intriguing difference between adult and neonatal kidneys is that the neonatal kidney has the capacity to control interstitial cell proliferation when the target number has been reached. In this study, we define the consequences of inactivating the TGFß/Smad response in the mouse interstitial cell lineage. We find that pathway inactivation through loss of Smad4 leads to overproliferation of interstitial cells regionally in the kidney medulla. Analysis of markers for BMP and TGFß pathway activation reveals that loss of Smad4 primarily reduces TGFß signaling in the interstitium. Whereas TGFß signaling is reduced in these cells, marker analysis shows that Wnt/ß-catenin signaling is increased. Our analysis supports a model in which Wnt/ß-catenin-mediated proliferation is attenuated by TGFß/Smad to ensure that proliferation ceases when the target number of interstitial cells has been reached in the neonatal medulla.

The Extracellular Matrix Environment of Clear Cell Renal Cell Carcinoma Determines Cancer Associated Fibroblast Growth.

Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer and is often caused by mutations in the oxygen-sensing machinery of kidney epithelial cells. Due to its pseudo-hypoxic state, ccRCC recruits extensive vasculature and other stromal components. Conventional cell culture methods provide poor representation of stromal cell types in primary cultures of ccRCC, and we hypothesized that mimicking the extracellular environment of the tumor would promote growth of both tumor and stromal cells. We employed proteomics to identify the components of ccRCC extracellular matrix (ECM) and found that in contrast to healthy kidney cortex, laminin, collagen IV, and entactin/nidogen are minor contributors. Instead, the ccRCC ECM is composed largely of collagen VI, fibronectin, and tenascin C. Analysis of single cell expression data indicates that cancer-associated fibroblasts are a major source of tumor ECM production. Tumor cells as well as stromal cells bind efficiently to a nine-component ECM blend characteristic of ccRCC. Primary patient-derived tumor cells bind the nine-component blend efficiently, allowing to us to establish mixed primary cultures of tumor cells and stromal cells. These miniature patient-specific replicas are conducive to microscopy and can be used to analyze interactions between cells in a model tumor microenvironment.

The Mesangial cell - the glomerular stromal cell.

Mesangial cells are stromal cells that are important for kidney glomerular homeostasis and the glomerular response to injury. A growing body of evidence demonstrates that mesenchymal stromal cells, such as stromal fibroblasts, pericytes and vascular smooth muscle cells, not only specify the architecture of tissues but also regulate developmental processes, vascularization and cell fate specification. In addition, through crosstalk with neighbouring cells and indirectly through the remodelling of the matrix, stromal cells can regulate a variety of processes such as immunity, inflammation, regeneration and in the context of maladaptive responses - fibrosis. Insights into the molecular phenotype of kidney mesangial cells suggest that they are a specialized stromal cell of the glomerulus. Here, we review our current understanding of mesenchymal stromal cells and discuss how it informs the function of mesangial cells and their role in disease. These new insights could lead to a better understanding of kidney disease pathogenesis and the development of new therapies for chronic kidney disease.

An efficient method to generate kidney organoids at the air-liquid interface.

The prevalence of kidney dysfunction continues to increase worldwide, driving the need to develop transplantable renal tissues. The kidney develops from four major renal progenitor populations: nephron epithelial, ureteric epithelial, interstitial and endothelial progenitors. Methods have been developed to generate kidney organoids but few or dispersed tubular clusters within the organoids hamper its use in regenerative applications. Here, we describe a detailed protocol of asynchronous mixing of kidney progenitors using organotypic culture conditions to generate kidney organoids tightly packed with tubular clusters and major renal structures including endothelial network and functional proximal tubules. This protocol provides guidance in the culture of human embryonic stem cells from a National Institute of Health-approved line and their directed differentiation into kidney organoids. Our 18-day protocol provides a rapid method to generate kidney organoids that facilitate the study of different nephrological events including in vitro tissue development, disease modeling and chemical screening. However, further studies are required to optimize the protocol to generate additional renal-specific cell types, interconnected nephron segments and physiologically functional renal tissues.

Development of a mechanically matched silk scaffolded 3D clear cell renal cell carcinoma model.

Development of a 3D, biomaterials-based model for clear cell renal cell carcinoma (ccRCC) would be advantageous for understanding disease progression in vitro. This study demonstrated the development of lyophilized silk scaffolds that mechanically match the experimentally determined Young's modulus for ex vivo ccRCC samples and normal kidney tissue. Scaffolds fabricated from silk solutions ranging from 3 to 12% (w/v) were evaluated through mechanical testing. Following mechanical characterization of ccRCC samples, it was demonstrated that 6% silk scaffolds mechanically matched ccRCC samples. No impact of pathological grade and stage on the calculated ccRCC modulus was observed and all tumors evaluated mechanically matched the 6% silk scaffold formulation. Stratifying tissue specimens based upon histological observations (e.g. evidence of high levels of collagen deposition) resulted in no significant differences between groups. To investigate the impact of a mechanically matched culturing environment on in vitro ccRCC disease characteristics a model ccRCC cell line, 786-O, was utilized. Scaffolded 786-O cells demonstrated increased lipid droplet accumulation, a hallmark of ccRCC, compared to standard two-dimensional (2D) culture conditions. Additionally, scaffolded 786-O cells demonstrated increased expression of genes associated with ccRCC aggressiveness (ex. VEGFA, TNF, and IL-6) or immune markers under investigation as therapeutic targets (ex. PDL1, CTLA4). Comparison with 786-O cells grown on non-mechanically matched scaffolds demonstrated that these improved ccRCC characteristics were driven by scaffold modulus. Overall, our findings support the use of silk scaffolds in replicating physiologic tumor behavior for clear cell renal cell carcinoma and provide a platform for investigating disease progression.

Vascular deficiencies in renal organoids and ex vivo kidney organogenesis.

Chronic kidney disease (CKD) and end stage renal disease (ESRD) are increasingly frequent and devastating conditions that have driven a surge in the need for kidney transplantation. A stark shortage of organs has fueled interest in generating viable replacement tissues ex vivo for transplantation. One promising approach has been self-organizing organoids, which mimic developmental processes and yield multicellular, organ-specific tissues. However, a recognized roadblock to this approach is that many organoid cell types fail to acquire full maturity and function. Here, we comprehensively assess the vasculature in two distinct kidney organoid models as well as in explanted embryonic kidneys. Using a variety of methods, we show that while organoids can develop a wide range of kidney cell types, as previously shown, endothelial cells (ECs) initially arise but then rapidly regress over time in culture. Vasculature of cultured embryonic kidneys exhibit similar regression. By contrast, engraftment of kidney organoids under the kidney capsule results in the formation of a stable, perfused vasculature that integrates into the organoid. This work demonstrates that kidney organoids offer a promising model system to define the complexities of vascular-nephron interactions, but the establishment and maintenance of a vascular network present unique challenges when grown ex vivo.

FOXD1 regulates cell division in clear cell renal cell carcinoma.

BACKGROUND: Forkhead transcription factors control cell growth in multiple cancer types. Foxd1 is essential for kidney development and mitochondrial metabolism, but its significance in renal cell carcinoma (ccRCC) has not been reported. METHODS: Transcriptome data from the TCGA database was used to correlate FOXD1 expression with patient survival. FOXD1 was knocked out in the 786-O cell line and known targets were analyzed. Reduced cell growth was observed and investigated in vitro using growth rate and Seahorse XF metabolic assays and in vivo using a xenograft model. Cell cycle characteristics were determined by flow cytometry and immunoblotting. Immunostaining for TUNEL and γH2AX was used to measure DNA damage. Association of the FOXD1 pathway with cell cycle progression was investigated through correlation analysis using the TCGA database. RESULTS: FOXD1 expression level in ccRCC correlated inversely with patient survival. Knockout of FOXD1 in 786-O cells altered expression of FOXD1 targets, particularly genes involved in metabolism (MICU1) and cell cycle progression. Investigation of metabolic state revealed significant alterations in mitochondrial metabolism and glycolysis, but no net change in energy production. In vitro growth rate assays showed a significant reduction in growth of 786-OFOXD1null. In vivo, xenografted 786-OFOXD1null showed reduced capacity for tumor formation and reduced tumor size. Cell cycle analysis showed that 786-OFOXD1null had an extended G2/M phase. Investigation of mitosis revealed a deficiency in phosphorylation of histone H3 in 786-OFOXD1null, and increased DNA damage. Genes correlate with FOXD1 in the TCGA dataset associate with several aspects of mitosis, including histone H3 phosphorylation. CONCLUSIONS: We show that FOXD1 regulates the cell cycle in ccRCC cells by control of histone H3 phosphorylation, and that FOXD1 expression governs tumor formation and tumor growth. Transcriptome analysis supports this role for FOXD1 in ccRCC patient tumors and provides an explanation for the inverse correlation between tumor expression of FOXD1 and patient survival. Our findings reveal an important role for FOXD1 in maintaining chromatin stability and promoting cell cycle progression and provide a new tool with which to study the biology of FOXD1 in ccRCC.

A ß-catenin-driven switch in TCF/LEF transcription factor binding to DNA target sites promotes commitment of mammalian nephron progenitor cells.

The canonical Wnt pathway transcriptional co-activator ß-catenin regulates self-renewal and differentiation of mammalian nephron progenitor cells (NPCs). We modulated ß-catenin levels in NPC cultures using the GSK3 inhibitor CHIR99021 (CHIR) to examine opposing developmental actions of ß-catenin. Low CHIR-mediated maintenance and expansion of NPCs are independent of direct engagement of TCF/LEF/ß-catenin transcriptional complexes at low CHIR-dependent cell-cycle targets. In contrast, in high CHIR, TCF7/LEF1/ß-catenin complexes replaced TCF7L1/TCF7L2 binding on enhancers of differentiation-promoting target genes. Chromosome confirmation studies showed pre-established promoter-enhancer connections to these target genes in NPCs. High CHIR-associated de novo looping was observed in positive transcriptional feedback regulation to the canonical Wnt pathway. Thus, ß-catenin's direct transcriptional role is restricted to the induction of NPCs, where rising ß-catenin levels switch inhibitory TCF7L1/TCF7L2 complexes to activating LEF1/TCF7 complexes at primed gene targets poised for rapid initiation of a nephrogenic program.

Asynchronous mixing of kidney progenitor cells potentiates nephrogenesis in organoids.

A fundamental challenge in emulating kidney tissue formation through directed differentiation of human pluripotent stem cells is that kidney development is iterative, and to reproduce the asynchronous mix of differentiation states found in the fetal kidney we combined cells differentiated at different times in the same organoid. Asynchronous mixing promoted nephrogenesis, and heterochronic organoids were well vascularized when engrafted under the kidney capsule. Micro-CT and injection of a circulating vascular marker demonstrated that engrafted kidney tissue was connected to the systemic circulation by 2 weeks after engraftment. Proximal tubule glucose uptake was confirmed, but despite these promising measures of graft function, overgrowth of stromal cells prevented long-term study. We propose that this is a technical feature of the engraftment procedure rather than a specific shortcoming of the directed differentiation because kidney organoids derived from primary cells and whole embryonic kidneys develop similar stromal overgrowth when engrafted under the kidney capsule.

Kidney-in-a-lymph node: A novel organogenesis assay to model human renal development and test nephron progenitor cell fates.

Stem cell-derived organoids are emerging as sophisticated models for studying development and disease and as potential sources for developing organ substitutes. Unfortunately, although organoids containing renal structures have been generated from mouse and human pluripotent stem cells, there are still critical unanswered questions that are difficult to attain via in vitro systems, including whether these nonvascularized organoids have a stable and physiologically relevant phenotype or whether a suitable transplantation site for long-term in vivo studies can be identified. Even orthotopic engraftment of organoid cultures in the adult does not provide an environment conducive to vascularization and functional differentiation. Previously, we showed that the lymph node offers an alternative transplantation site where mouse metanephroi can differentiate into mature renal structures with excretory, homeostatic, and endocrine functions. Here, we show that the lymph node lends itself well as a niche to also grow human primary kidney rudiments and can additionally be viewed as a platform to interrogate emerging renal organoid cultures. Our study has a wide-ranging impact for tissue engineering approaches to rebuild functional tissues in vivo including-but not limited to-the kidney.

Scaffolding kidney organoids on silk.

End stage kidney disease affects hundreds of thousands of patients in the United States. The therapy of choice is kidney replacement, but availability of organs is limited, and alternative sources of tissue are needed. Generation of new kidney tissue in the laboratory has been made possible through pluripotent cell reprogramming and directed differentiation. In current procedures, aggregates of cells known as organoids are grown either submerged or at the air-liquid interface. These studies have demonstrated that kidney tissue can be generated from pluripotent stem cells, but they also identify limitations. The first is that perfusion of cell aggregates is limited, restricting the size to which they can be grown. The second is that aggregates lack the structural integrity required for convenient engraftment and suturing or adhesion to regions of kidney injury. In this study, we evaluated the capacity of silk to serve as a support for the growth and differentiation of kidney tissue from primary cells and from human induced pluripotent stem cells. We find that cells can differentiate to epithelia characteristic of the developing kidney on this material and that these structures are maintained following engraftment under the capsule of the adult kidney. Blood vessel investment can be promoted by the addition of vascular endothelial growth factor to the scaffold, but the proliferation of stromal cells within the graft presents a challenge, which will require some readjustment of cell growth and differentiation conditions. In summary, we find that silk can be used to support growth of stem cell derived kidney tissue.

Long-Term Culture of Nephron Progenitor Cells Ex Vivo.

Nephrons differentiate from the cap mesenchyme of the fetal kidney. Nephron progenitor cells that populate the cap mesenchyme efficiently balance self-renewal and epithelial differentiation to enable repeated rounds of nephron formation during development. Here we describe a method to isolate and propagate these cells from the embryonic mouse kidney. Using this method, nephron progenitor cells from a single litter of mice can be propagated to hundreds of millions of cells that express appropriate markers of the undifferentiated state and retain epithelial differentiation capacity in vitro.

Kidney Nephron Determination.

The nephron is a multifunctional filtration device equipped with an array of sophisticated sensors. For appropriate physiological function in the human and mouse, nephrons must be stereotypically arrayed in large numbers, and this essential structural property that defines the kidney is determined during its fetal development. This review explores the process of nephron determination in the fetal kidney, providing an overview of the foundational literature in the field as well as exploring new developments in this dynamic research area. Mechanisms that ensure that a large number of nephrons can be formed from a small initial number of progenitor cells are central to this process, and the question of how the nephron progenitor cell population balances epithelial differentiation with renewal in the progenitor state is a subject of particular interest. Key growth factor signaling pathways and transcription factor networks are discussed.