A comparative study of cellular diversity between the Xenopus pronephric and mouse metanephric nephron.

The kidney is an essential organ that ensures bodily fluid homeostasis and removes soluble waste products from the organism. Nephrons, the functional units of the kidney, comprise a blood filter, the glomerulus or glomus, and an epithelial tubule that processes the filtrate from the blood or coelom and selectively reabsorbs solutes, such as sugars, proteins, ions, and water, leaving waste products to be eliminated in the urine. Genes coding for transporters are segmentally expressed, enabling the nephron to sequentially process the filtrate. The Xenopus embryonic kidney, the pronephros, which consists of a single large nephron, has served as a valuable model to identify genes involved in nephron formation and patterning. Therefore, the developmental patterning program that generates these segments is of great interest. Prior work has defined the gene expression profiles of Xenopus nephron segments via in situ hybridization strategies, but a comprehensive understanding of the cellular makeup of the pronephric kidney remains incomplete. Here, we carried out single-cell mRNA sequencing of the functional Xenopus pronephric nephron and evaluated its cellular composition through comparative analyses with previous Xenopus studies and single-cell mRNA sequencing of the adult mouse kidney. This study reconstructs the cellular makeup of the pronephric kidney and identifies conserved cells, segments, and associated gene expression profiles. Thus, our data highlight significant conservation in podocytes, proximal and distal tubule cells, and divergence in cellular composition underlying the capacity of each nephron to remove wastes in the form of urine, while emphasizing the Xenopus pronephros as a model for physiology and disease.

Tissue-Targeted CRISPR-Cas9-Mediated Genome Editing of Multiple Homeologs in F0-Generation Xenopus laevis Embryos.

Xenopus laevis frogs are a powerful developmental model that enables studies combining classical embryology and molecular manipulation. Because of the large embryo size, ease of microinjection, and ability to target tissues through established fate maps, X. laevis has become the predominant amphibian research model. Given that their allotetraploid genome has complicated the generation of gene knockouts, strategies need to be established for efficient mutagenesis of multiple homeologs to evaluate gene function. Here we describe a protocol to use CRISPR-Cas9-mediated genome editing to target either single alleles or multiple alloalleles in F0 X. laevis embryos. A single-guide RNA (sgRNA) is designed to target a specific DNA sequence encoding a critical protein domain. To mutagenize a gene with two alloalleles, the sgRNA is designed against a sequence that is common to both homeologs. This sgRNA, along with the Cas9 protein, is microinjected into the zygote to disrupt the genomic sequences in the whole embryo or into a specific blastomere for tissue-targeted effects. Error-prone repair of CRISPR-Cas9-generated DNA double-strand breaks leads to insertions and deletions creating mosaic gene lesions within the embryos. The genomic DNA isolated from each mosaic F0 embryo is sequenced, and software is applied to assess the nature of the mutations generated and degree of mosaicism. This protocol enables the knockout of genes within the whole embryo or in specific tissues in F0 X. laevis embryos to facilitate the evaluation of resulting phenotypes.

The Wnt/PCP formin Daam1 drives cell-cell adhesion during nephron development.

E-cadherin junctions facilitate assembly and disassembly of cell contacts that drive development and homeostasis of epithelial tissues. In this study, using Xenopus embryonic kidney and Madin-Darby canine kidney (MDCK) cells, we investigate the role of the Wnt/planar cell polarity (PCP) formin Daam1 (Dishevelled-associated activator of morphogenesis 1) in regulating E-cadherin-based intercellular adhesion. Using live imaging, we show that Daam1 localizes to newly formed cell contacts in the developing nephron. Furthermore, analyses of junctional filamentous actin (F-actin) upon Daam1 depletion indicate decreased microfilament localization and slowed turnover. We also show that Daam1 is necessary for efficient and timely localization of junctional E-cadherin, mediated by Daam1's formin homology domain 2 (FH2). Finally, we establish that Daam1 signaling promotes organized movement of renal cells. This study demonstrates that Daam1 formin junctional activity is critical for epithelial tissue organization.

A catenin of the plakophilin-subfamily, Pkp3, responds to canonical-Wnt pathway components and signals.

Vertebrate beta-catenin plays a key role as a transducer of canonical-Wnt signals. We earlier reported that, similar to beta-catenin, the cytoplasmic signaling pool of p120-catenin-isoform1 is stabilized in response to canonical-Wnt signals. To obtain a yet broader view of the Wnt-pathway's impact upon catenin proteins, we focused upon plakophilin3 (plakophilin-3; Pkp3) as a representative of the plakophilin-catenin subfamily. Promoting tissue integrity, the plakophilins assist in linking desmosomal cadherins to intermediate filaments at desmosome junctions, and in common with other catenins they perform additional functions including in the nucleus. In this report, we test whether canonical-Wnt pathway components modulate Pkp3 protein levels. We find that in common with beta-catenin and p120-catenin-isoform1, Pkp3 is stabilized in the presence of a Wnt-ligand or a dominant-active form of the LRP6 receptor. Pkp3's levels are conversely lowered upon expressing destruction-complex components such as GSK3ß and Axin, and in further likeness to beta-catenin and p120-isoform1, Pkp3 associates with GSK3beta and Axin. Finally, we note that Pkp3-catenin trans-localizes into the nucleus in response to Wnt-ligand and its exogenous expression stimulates an accepted Wnt reporter. These findings fit an expanded model where context-dependent Wnt-signals or pathway components modulate Pkp3-catenin levels. Future studies will be needed to assess potential gene regulatory, cell adhesive, or cytoskeletal effects.

Aquatic models of human ciliary diseases.

Cilia are microtubule-based structures that either transmit information into the cell or move fluid outside of the cell. There are many human diseases that arise from malfunctioning cilia. Although mammalian models provide vital insights into the underlying pathology of these diseases, aquatic organisms such as Xenopus and zebrafish provide valuable tools to help screen and dissect out the underlying causes of these diseases. In this review we focus on recent studies that identify or describe different types of human ciliopathies and outline how aquatic organisms have aided our understanding of these diseases.

Interprofessional 30-day readmission review novel curriculum.

Planning and coordination among health-care professionals decrease readmission rates, yet workers have few opportunities to learn interprofessionally to improve transitions of care. An interprofessional readmission review curriculum engaged medical residents, pharmacy residents, nurse practitioner students, early-career nurses, and social work students in a critical analysis of readmissions. Learners (N = 98) participated in a 2 h, collaborative learning session to review health records from a patient readmitted within 30 days of discharge and determine plausible root causes for readmissions. A 5-item post-session survey completed by 83 (85%) evaluated knowledge and perceived competencies in transitions of care before and after participation. Significant improvements (p < .001) occurred in ratings for all five items. Two open-ended questions captured learners' perceptions of understanding and appreciating the roles of other disciplines in the discharge process and importance of interprofessional communication. Several themes emerged including understanding gaps in the discharge process, improving interprofessional collaboration and communication, and paying more attention to discharge documentation. This innovative program helped build essential skills to ensure safe discharges by introducing learners to interprofessional perspectives in analyzing root causes for readmissions, strategies to improve discharge planning, and the value of team-based care.

Home-Based Medical Care: High-Value Health Care During Coronavirus Disease 2019 and Beyond.

Ms. H is a 78-year-old woman with a history of congestive heart failure, chronic obstructive pulmonary disease, and recent stroke who was discharged 1 month ago from a subacute rehabilitation facility. She moved in with her son because she now requires a walker and cannot return to her third-floor apartment. One evening, Ms. H develops a low-grade fever and mild shortness of breath intermittently relieved by her albuterol inhaler. Her son is worried, but knows that his mom does not want to return to the hospital.

Overview of High Yield Geriatrics Assessment for Clinic and Hospital.

Geriatric assessment is a comprehensive, multifaceted, and interdisciplinary evaluation of medical, socioeconomic, environmental, and functional concerns unique to older adults; it can be focused or broadened according to the needs of the patient and the concerns of clinical providers. Herein, the authors present a high-yield framework that can be used to assess older adult patients across a variety of settings.

Correction: DYRK1A-related intellectual disability: a syndrome associated with congenital anomalies of the kidney and urinary tract.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Divergent roles of the Wnt/PCP Formin Daam1 in renal ciliogenesis.

Kidneys are composed of numerous ciliated epithelial tubules called nephrons. Each nephron functions to reabsorb nutrients and concentrate waste products into urine. Defects in primary cilia are associated with abnormal formation of nephrons and cyst formation in a wide range of kidney disorders. Previous work in Xenopus laevis and zebrafish embryos established that loss of components that make up the Wnt/PCP pathway, Daam1 and ArhGEF19 (wGEF) perturb kidney tubulogenesis. Dishevelled, which activates both the canonical and non-canonical Wnt/PCP pathway, affect cilia formation in multiciliated cells. In this study, we investigated the role of the noncanoncial Wnt/PCP components Daam1 and ArhGEF19 (wGEF) in renal ciliogenesis utilizing polarized mammalian kidney epithelia cells (MDCKII and IMCD3) and Xenopus laevis embryonic kidney. We demonstrate that knockdown of Daam1 and ArhGEF19 in MDCKII and IMCD3 cells leads to loss of cilia, and Daam1's effect on ciliogenesis is mediated by the formin-activity of Daam1. Moreover, Daam1 co-localizes with the ciliary transport protein Ift88 and is present in cilia. Interestingly, knocking down Daam1 in Xenopus kidney does not lead to loss of cilia. These data suggests a new role for Daam1 in the formation of primary cilia.

DYRK1A-related intellectual disability: a syndrome associated with congenital anomalies of the kidney and urinary tract.

PURPOSE: Haploinsufficiency of DYRK1A causes a recognizable clinical syndrome. The goal of this paper is to investigate congenital anomalies of the kidney and urinary tract (CAKUT) and genital defects (GD) in patients with DYRK1A variants. METHODS: A large database of clinical exome sequencing (ES) was queried for de novo DYRK1A variants and CAKUT/GD phenotypes were characterized. Xenopus laevis (frog) was chosen as a model organism to assess Dyrk1a's role in renal development. RESULTS: Phenotypic details and variants of 19 patients were compiled after an initial observation that one patient with a de novo pathogenic variant in DYRK1A had GD. CAKUT/GD data were available from 15 patients, 11 of whom presented with CAKUT/GD. Studies in Xenopus embryos demonstrated that knockdown of Dyrk1a, which is expressed in forming nephrons, disrupts the development of segments of embryonic nephrons, which ultimately give rise to the entire genitourinary (GU) tract. These defects could be rescued by coinjecting wild-type human DYRK1A RNA, but not with DYRK1AR205* or DYRK1AL245R RNA. CONCLUSION: Evidence supports routine GU screening of all individuals with de novo DYRK1A pathogenic variants to ensure optimized clinical management. Collectively, the reported clinical data and loss-of-function studies in Xenopus substantiate a novel role for DYRK1A in GU development.

Modeling congenital kidney diseases in Xenopus laevis.

Congenital anomalies of the kidney and urinary tract (CAKUT) occur in ∼1/500 live births and are a leading cause of pediatric kidney failure. With an average wait time of 3-5 years for a kidney transplant, the need is high for the development of new strategies aimed at reducing the incidence of CAKUT and preserving renal function. Next-generation sequencing has uncovered a significant number of putative causal genes, but a simple and efficient model system to examine the function of CAKUT genes is needed. Xenopus laevis (frog) embryos are well-suited to model congenital kidney diseases and to explore the mechanisms that cause these developmental defects. Xenopus has many advantages for studying the kidney: the embryos develop externally and are easily manipulated with microinjections, they have a functional kidney in ∼2 days, and 79% of identified human disease genes have a verified ortholog in Xenopus This facilitates high-throughput screening of candidate CAKUT-causing genes. In this Review, we present the similarities between Xenopus and mammalian kidneys, highlight studies of CAKUT-causing genes in Xenopus and describe how common kidney diseases have been modeled successfully in this model organism. Additionally, we discuss several molecular pathways associated with kidney disease that have been studied in Xenopus and demonstrate why it is a useful model for studying human kidney diseases.

Dynamin Binding Protein Is Required for Xenopus laevis Kidney Development.

The adult human kidney contains over one million nephrons, with each nephron consisting of a tube containing segments that have specialized functions in nutrient and water absorption and waste excretion. The embryonic kidney of Xenopus laevis consists of a single functional nephron composed of regions that are analogous to those found in the human nephron, making it a simple model for the study of nephrogenesis. The exocyst complex, which traffics proteins to the cell membrane in vesicles via CDC42, is essential for normal kidney development. Here, we show that the CDC42-GEF, dynamin binding protein (Dnmbp/Tuba), is essential for nephrogenesis in Xenopus. dnmbp is expressed in Xenopus embryo kidneys during development, and knockdown of Dnmbp using two separate morpholino antisense oligonucleotides results in reduced expression of late pronephric markers, whereas the expression of early markers of nephrogenesis remains unchanged. A greater reduction in expression of markers of differentiated distal and connecting tubules was seen in comparison to proximal tubule markers, indicating that Dnmbp reduction may have a greater impact on distal and connecting tubule differentiation. Additionally, Dnmbp reduction results in glomus and ciliary defects. dnmbp knockout using CRISPR results in a similar reduction of late markers of pronephric tubulogenesis and also results in edema formation in later stage embryos. Overexpression of dnmbp in the kidney also resulted in disrupted pronephric tubules, suggesting that dnmbp levels in the developing kidney are tightly regulated, with either increased or decreased levels leading to developmental defects. Together, these data suggest that Dnmbp is required for nephrogenesis.