Temporally and Spatially Regulated Collagen XVIII Isoforms Impact Ureteric Patterning Through Their TSP1-like Domain

ABSTRACTCollagen XVIII (ColXVIII) is a component of the extracellular matrix implicated in embryogenesis and control of homeostasis. We provide evidence that ColXVIII has a specific role in kidney ontogenesis by regulating the interaction between mesenchymal and epithelial tissues as observed in analyses of total and isoform-specific knockout embryos, mice, andex vivoorgan primordia. ColXVIII deficiency, both temporally and spatially, impacts the 3D pattern of ureteric tree branching morphogenesisviaits specific isoforms. Proper development of ureteric tree depends on a tight control of the nephron progenitor cells (NPCs). ColXVIII-deficient NPCs are leaving the NPC pool faster than in controls. Moreover, the data suggests that ColXVIII mediates the kidney epithelial tree patterningviaits N-terminal domains, and especially the Thrombospondin-1-like domain, and that this morphogenetic effect involves ureteric epithelial integrins. Altogether, the results propose a significant role for ColXVIII in a complex signalling network regulating renal progenitors and kidney development.

Molecular Mechanisms of Renal Progenitor Regulation: How Many Pieces in the Puzzle?

Kidneys of mice, rats and humans possess progenitors that maintain daily homeostasis and take part in endogenous regenerative processes following injury, owing to their capacity to proliferate and differentiate. In the glomerular and tubular compartments of the nephron, consistent studies demonstrated that well-characterized, distinct populations of progenitor cells, localized in the parietal epithelium of Bowman capsule and scattered in the proximal and distal tubules, could generate segment-specific cells in physiological conditions and following tissue injury. However, defective or abnormal regenerative responses of these progenitors can contribute to pathologic conditions. The molecular characteristics of renal progenitors have been extensively studied, revealing that numerous classical and evolutionarily conserved pathways, such as Notch or Wnt/β-catenin, play a major role in cell regulation. Others, such as retinoic acid, renin-angiotensin-aldosterone system, TLR2 (Toll-like receptor 2) and leptin, are also important in this process. In this review, we summarize the plethora of molecular mechanisms directing renal progenitor responses during homeostasis and following kidney injury. Finally, we will explore how single-cell RNA sequencing could bring the characterization of renal progenitors to the next level, while knowing their molecular signature is gaining relevance in the clinic.

Molecular Mechanisms of Renal Progenitor Regulation: How Many Pieces in the Puzzle?

Kidneys of mice, rats and humans possess progenitors that maintain daily homeostasis and take part in endogenous regenerative processes following injury, owing to their capacity to proliferate and differentiate. In the glomerular and tubular compartments of the nephron, consistent studies demonstrated that well-characterized, distinct populations of progenitor cells, localized in the parietal epithelium of Bowman capsule and scattered in the proximal and distal tubules, could generate segment-specific cells in physiological conditions and following tissue injury. However, defective or abnormal regenerative responses of these progenitors can contribute to pathologic conditions. The molecular characteristics of renal progenitors have been extensively studied, revealing that numerous classical and evolutionarily conserved pathways, such as Notch or Wnt/β-catenin, play a major role in cell regulation. Others, such as retinoic acid, renin-angiotensin-aldosterone system, TLR2 (Toll-Like Receptor 2) and leptin, are also important in this process. In this review, we summarize the plethora of molecular mechanisms directing renal progenitor responses during homeostasis and following kidney injury. Finally, we will explore how single cell RNA sequencing could bring the characterization of renal progenitors to the next level, while knowing their molecular signature is gaining relevance in the clinic.

osr1 maintains renal progenitors and regulates podocyte development by promoting wnt2ba through antagonism of hand2

ABSTRACTKnowledge about the genetic pathways that control renal cell lineage development is essential to better understand the basis of congenital malformations of the kidney and design regenerative medicine therapies. The embryonic zebrafish kidney, or pronephros, contains two nephrons that are conserved with humans. Recently, the transcription factors Osr1 and Hand2 were found to exert antagonistic influences to balance kidney specification (Perens et al., 2016). Here, we performed a forward genetic screen in zebrafish to identify nephrogenesis regulators, where whole genome sequencing of the novel oceanside (ocn) mutant revealed a nonsense mutation in osr1. ocn mutants evince severe pronephros defects including abrogation of podocytes and proximal tubule cells. Our studies reveal that osr1 is not needed to specify renal progenitors, but rather required to maintain their survival. Additionally, osr1 is requisite for expression of the canonical Wnt ligand wnt2ba, where wnt2ba is expressed in the intermediate mesoderm (IM) and later restricts to podocytes. Deficiency of wnt2ba reduced podocyte progenitors, where overexpression of wnt2ba was sufficient to rescue the podocyte lineage as well as osr1 loss of function. Finally, we demonstrate that reciprocal antagonism between osr1 and hand2 mediates podocyte development specifically by controlling wnt2ba expression in the IM. Together, our data show that Osr1 is essential for a sequence of temporal functions that mediate the survival and lineage decisions of IM progenitors, and subsequently the maintenance of podocytes and proximal tubule epithelium in the embryonic nephron.

PKHhigh/CD133+/CD24− Renal Stem-Like Cells Isolated from Human Nephrospheres Exhibit In Vitro Multipotency

The mechanism upon which human kidneys undergo regeneration is debated, though different lineage-tracing mouse models have tried to explain the cellular types and the mechanisms involved. Different sources of human renal progenitors have been proposed, but it is difficult to argue whether these populations have the same capacities that have been described in mice. Using the nephrosphere (NS) model, we isolated the quiescent population of adult human renal stem-like PKHhigh/CD133+/CD24− cells (RSC). The aim of this study was to deepen the RSC in vitro multipotency capacity. RSC, not expressing endothelial markers, generated secondary nephrospheres containing CD31+/vWf+ cells and cytokeratin positive cells, indicating the coexistence of endothelial and epithelial commitment. RSC cultured on decellularized human renal scaffolds generated endothelial structures together with the proximal and distal tubular structures. CD31+ endothelial committed progenitors sorted from nephrospheres generated spheroids with endothelial-like sprouts in Matrigel. We also demonstrated the double commitment toward endothelial and epithelial lineages of single RSC. The ability of the plastic RSC population to recapitulate the development of tubular epithelial and endothelial renal lineages makes these cells a good tool for the creation of organoids with translational relevance for studying the parenchymal and endothelial cell interactions and developing new therapeutic strategies.

MO060ACUTE KIDNEY INJURY PROMOTES DEVELOPMENT OF A PAPILLARY RENAL CELL ADENOMA-CARCINOMA SEQUENCE FROM RENAL PROGENITORS

Background and Aims Renal cell carcinoma (RCC) accounts for 2% of all cancers, with about 190,000 new cases per year worldwide. Risk factors for RCC include obesity, diabetes, hypertension and genetic factors, but the majority of cancers occur in apparent absence of clear risk factors. Acute tissue injury (AKI) causes DNA damage and repair processes involving increased cell mitosis and polyploidization, leading to cell function alterations that may potentially drive cancer development. We proposed to verify whether AKI plays a role in RCC development, and to identify the cellular origin of RCC. Method We used the following techniques: 1. observational, retrospective clinical trial to identify a possible association of AKI with RCC. 2. Experimental AKI induction in wild-type mice to study tumor development over 36 weeks. 3. Analysis of TCGA Research Network dataset on human papillary RCC (pRCC) molecular characterization, focusing on AKI-driven pathways. 4. Development of mouse models in which the intracellular domain of Notch 1 (NICD1), a molecule modulated during AKI, is expressed constitutively by all Pax8+ tubular epithelial cells (Pax8/NICD1) or only by Pax2+ renal progenitors (Pax2/NICD1) upon induction in adult mice. The mice were sacrificed at 36 weeks or 4 weeks after AKI. 5. Clonal analysis of tumoral lesions with Confetti reporter. 6. Examination of single cell RNA sequencing (RNAseq) data from pRCC patients. Results We observed that an AKI episode is a major risk factor for pRCC development and recurrence in patients. Wild-type mice subjected to AKI developed pRCC over time in an adenoma-carcinoma sequence, corroborating our human findings. Among AKI-related pathways, Notch1 overexpression in human pRCC associated with worse outcome, prompting us to generate Notch1-overexpressing mice. At 36 weeks o at 4 weeks following AKI, Pax8/NICD1 mice presented a significant decline of renal excretory function as well as type 2 pRCCs. Confetti lineage tracing showed that most of the pRCCs were monoclonal or biclonal, suggesting that they could originate from a local stem cell/progenitor population. Pax2/NICD1 mice presented type 2 pRCCs, and lineage tracing identified single Pax2+ tubular progenitors as the source of pRCCs. Single cell RNAseq analysis confirmed that the molecular signature of the pRCC cell of origin matched the one of human tubular progenitors. Conclusion This study expose the link between AKI and pRCC development in patients, with important clinical implications. In mice, AKI promotes long-term development of type 2 papillary tumors by activating the AKI-associated Notch1 pathway. Additionally, pRCC originates from clonal proliferation of renal progenitors in a classical adenoma-carcinoma sequence leading to invasive pRCC growth and metastatization in mice.

Kidney Cells Regeneration: Dedifferentiation of Tubular Epithelium, Resident Stem Cells and Possible Niches for Renal Progenitors

A kidney is an organ with relatively low basal cellular regenerative potential. However, renal cells have a pronounced ability to proliferate after injury, which undermines that the kidney cells are able to regenerate under induced conditions. The majority of studies explain yielded regeneration either by the dedifferentiation of the mature tubular epithelium or by the presence of a resident pool of progenitor cells in the kidney tissue. Whether cells responsible for the regeneration of the kidney initially have progenitor properties or if they obtain a “progenitor phenotype” during dedifferentiation after an injury, still stays the open question. The major stumbling block in resolving the issue is the lack of specific methods for distinguishing between dedifferentiated cells and resident progenitor cells. Transgenic animals, single-cell transcriptomics, and other recent approaches could be powerful tools to solve this problem. This review examines the main mechanisms of kidney regeneration: dedifferentiation of epithelial cells and activation of progenitor cells with special attention to potential niches of kidney progenitor cells. We attempted to give a detailed description of the most controversial topics in this field and ways to resolve these issues.

Paracrine and autocrine R-spondin signalling is essential for the maintenance and differentiation of renal stem cells

During kidney development, WNT/β-catenin signalling has to be tightly controlled to ensure proliferation and differentiation of renal stem cells. Here we show that the two signalling molecules RSPO1 and RSPO3 act in a functionally redundant manner to permit WNT/β-catenin signalling and their genetic deletion leads to a rapid decline of renal progenitors. By contrast, tissue specific deletion in cap mesenchymal cells abolishes mesenchyme to epithelial transition (MET) that is linked to a loss of Bmp7 expression, absence of SMAD1/5 phosphorylation and a concomitant failure to activate Lef1, Fgf8 and Wnt4, thus explaining the observed phenotype on a molecular level. Surprisingly, the full knockout of LGR4/5/6, the cognate receptors of R-spondins, only mildly affects progenitor numbers, but does not interfere with MET. Taken together our data demonstrate key roles for R-spondins in permitting stem cell maintenance and differentiation and reveal Lgr-dependent and independent functions for these ligands during kidney formation.

Molecular Mechanisms of the Acute Kidney Injury to Chronic Kidney Disease Transition: An Updated View

Increasing evidence has demonstrated the bidirectional link between acute kidney injury (AKI) and chronic kidney disease (CKD) such that, in the clinical setting, the new concept of a unified syndrome has been proposed. The pathophysiological reasons, along with the cellular and molecular mechanisms, behind the ability of a single, acute, apparently self-limiting event to drive chronic kidney disease progression are yet to be explained. This acute injury could promote progression to chronic disease through different pathways involving the endothelium, the inflammatory response and the development of fibrosis. The interplay among endothelial cells, macrophages and other immune cells, pericytes and fibroblasts often converge in the tubular epithelial cells that play a central role. Recent evidence has strengthened this concept by demonstrating that injured tubules respond to acute tubular necrosis through two main mechanisms: The polyploidization of tubular cells and the proliferation of a small population of self-renewing renal progenitors. This alternative pathophysiological interpretation could better characterize functional recovery after AKI.