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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.

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.Mesangial cells are stromal cells that are important for kidney glomerular homeostasis and the glomerular response to injury. This Perspective reviews advances in our understanding of mesenchymal stromal cell function and describes how these insights can inform our understanding of mesangial cells and their role in disease.

The canonical Wnt pathway transcriptional co-activator β-catenin regulates self-renewal and differentiation of mammalian nephron progenitor cells (NPCs). We modulated β-catenin levels in NPC cultures using the GSK3 inhibitor CHIR99021 (CHIR) to examine opposing developmental actions of β-catenin. Low CHIR-mediated maintenance and expansion of NPCs are independent of direct engagement of TCF/LEF/β-catenin transcriptional complexes at low CHIR-dependent cell-cycle targets. In contrast, in high CHIR, TCF7/LEF1/β-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, β-catenin’s direct transcriptional role is restricted to the induction of NPCs, where rising β-catenin levels switch inhibitory TCF7L1/TCF7L2 complexes to activating LEF1/TCF7 complexes at primed gene targets poised for rapid initiation of a nephrogenic program.

The proximal tubule of the nephron performs energy-demanding functions such as resorption of water, amino acids and glucose. Formation of the energy-producing machinery is an essential step in proximal tubule epithelial cell differentiation, and this report asks how mitochondria are localized within these cells. We show that mitochondria move from the apical to basolateral side of the proximal tubule cell coincident with the initiation of lumen flow and that proximal tubules deficient in filtration maintain mitochondria in the apical position. Mitochondrial localization depends on the activity of LRRK2 and modeling fluid flow on cultured proximal tubule epithelial cells demonstrates that LRRK2 activity is regulated by fluid shear stress, explaining how onset of flow in the newly differentiated proximal tubule may trigger the apical-to-basolateral dissemination of mitochondria. These findings indicate that mitochondrial redistribution is one component of a cellular program in the nascent proximal tubule that drives function and that this process is triggered by flow. Formation of the energy-producing machinery in the proximal tubule of the nephron is an essential step in differentiation. The authors show that mitochondrial localization depends on LRRK2, the activity of which is modulated by fluid flow.

Embryonic nephron progenitor cells are segregated in molecularly distinct compartments of unknown function. Our study reveals an integral role for bone morphogenetic protein-SMAD in promoting transition of progenitors from the primitive Cbp/p300-interacting transactivator 1 expressing (CITED1+) compartment to the uniquely sine oculis-related homeobox 2 expressing (SIX2-only) compartment where they become inducible by wingless-type mouse mammary tumor virus integration site family member (WNT)/β-catenin signaling. Significantly, CITED1 ⁺ cells are refractory to WNT/β-catenin induction. We propose a model in which the primitive CITED1 ⁺ compartment is refractory to induction by WNT9b/β-catenin, ensuring maintenance of undifferentiated progenitor cells for future nephrogenesis. Bone morphogenetic protein 7-SMAD is then required for transition to a distinct compartment in which cells become inducible by WNT9b/β-catenin, allowing them to progress toward epithelialization.

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-O FOXD1null . In vivo, xenografted 786-O FOXD1null showed reduced capacity for tumor formation and reduced tumor size. Cell cycle analysis showed that 786-O FOXD1null had an extended G2/M phase. Investigation of mitosis revealed a deficiency in phosphorylation of histone H3 in 786-O FOXD1null , 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.

The different segments of the nephron and glomerulus in the kidney balance the processes of water homeostasis, solute recovery, blood filtration, and metabolite excretion. When segment function is disrupted, a range of pathological features are presented. Little is known about nephron patterning during embryogenesis. In this study, we demonstrate that the early nephron is patterned by a gradient in β-catenin activity along the axis of the nephron tubule. By modifying β-catenin activity, we force cells within nephrons to differentiate according to the imposed β-catenin activity level, thereby causing spatial shifts in nephron segments. The β-catenin signalling gradient interacts with the BMP pathway which, through PTEN/PI3K/AKT signalling, antagonises β-catenin activity and promotes segment identities associated with low β-catenin activity. β-catenin activity and PI3K signalling also integrate with Notch signalling to control segmentation: modulating β-catenin activity or PI3K rescues segment identities normally lost by inhibition of Notch. Our data therefore identifies a molecular network for nephron patterning. The main function of the kidney is to filter blood to remove waste and regulate the amount of water and salt in the body. Structures in the kidney—called nephrons—do much of this work and blood is filtered in a part of each nephron called the glomerulus. The substances filtered out of the blood move into a series of ‘tubules’, another part of the nephrons, from where water and soluble substances are reabsorbed or excreted as the body requires. If the nephrons do not work correctly, it can lead to a wide range of health problems—from abnormal water and salt loss to dangerously high blood pressure. For organs and tissues to develop in an embryo, signalling pathways help cells to communicate with each other. These pathways control what type of cells the embryonic cells become and also help neighbouring cells work together to form specialised structures with particular functions. Much is unknown about how the nephron develops, including how its different structures coordinate their development with each other so that they form in the right position in the nephron. A protein called beta-catenin was already known to play an important role in the signalling pathways that trigger the earliest stages of nephron formation. Lindström et al. further investigated how this protein helps the nephron to develop by using a wide range of techniques, including growing genetically altered mouse kidneys in culture and capturing images of the developing nephrons with time-lapse microscopy. The combined results reveal that the levels of beta-catenin activity coordinate the development of the different structures in the nephron. The beta-catenin protein is not equally active in all parts of the nephron; instead, it forms a gradient of different activity levels. The highest levels of beta-catenin activity occur in the tubules at the furthest end of the developing nephron; this activity gradually decreases along the length of the nephron, and the glomerulus itself lacks beta-catenin activity altogether. Experimentally manipulating the levels of beta-catenin at different points along the nephron caused those cells to take on the wrong identity, causing parts of the nephron to form in the wrong place. Lindström et al. were also able to establish that the signalling pathway controlled by beta-catenin activity interacts with three other well-known signalling pathways as part of a network that controls nephron development. More research is required to find out which signal activates beta-catenin in the first place and from where in the kidney this signal comes. It also remains to be discovered how a particular cell in the tubule interprets the exact activities of the different signals to give the cell its specific identity for that place in the nephron. A better understanding of these sorts of processes will eventually help build new kidneys for people with kidney failure.

The final nephron number in an adult kidney is regulated by nephron progenitor cell availability and collecting duct branching in the fetal period. Fetal environmental perturbations that cause reductions in cell numbers in these two compartments result in low nephron endowment. Previous work has shown that maternal dietary factors influence nephron progenitor cell availability, with both caloric restriction and protein deprivation leading to reduced cell numbers through apoptosis. In this study, we evaluate the consequences of inducing nephron progenitor cell death on progenitor niche dynamics and on nephron endowment. Depletion of approximately 40% of nephron progenitor cells by expression of diphtheria toxin A at embryonic day 15 in the mouse results in 10-20% nephron reduction in the neonatal period. Analysis of cell numbers within the progenitor cell pool following induction of apoptosis reveals a compensatory response in which surviving progenitor cells increase their proliferation and replenish the niche. The proliferative response is temporally associated with infiltration of macrophages into the nephrogenic zone. Colony stimulating factor 1 (CSF1) has a mitogenic effect on nephron progenitor cells, providing a potential explanation for the compensatory proliferation. However, CSF1 also promotes interstitial cell proliferation, and the compensatory response is associated with interstitial expansion in recovering kidneys which can be pharmacologically inhibited by treatment with clodronate liposomes. Our findings suggest that the fetal kidney employs a macrophage-dependent compensatory regenerative mechanism to respond to acute injury caused by death of nephron progenitor cells, but that this regenerative response is associated with neonatal interstitial expansion.

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.