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.

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.

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.

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.

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.

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.

An Interactive Ambulatory Nephrology Curriculum for Internal Medicine Interns: Design, Implementation, and Participant Feedback.

While diminishing nephrology fellow recruitment is a known issue, more work is needed to evaluate possible interventions to reverse this trend. We designed and implemented a curriculum to increase exposure to ambulatory nephrology among internal medicine interns. The curriculum focused on key aspects of outpatient nephrology practice, including supervised clinic visits, formal themed didactic content, and an online interactive forum with assigned evidence-based readings and small-group responses to relevant cases. We obtained postcourse surveys from all participating interns. Of the 43 interns who took part in the first year of the ambulatory nephrology curriculum, 100% reported a positive didactic experience and 91% reported a positive interactive online experience. 77% reported an improvement in their familiarity with clinical nephrology practice (median 2-point increase in familiarity score on a 7-point scale, P<0.001 by signed rank testing). Qualitative feedback included praise for the high-yield topics covered by the lectures and energizing teachers. In conclusion, we successfully implemented an ambulatory nephrology curriculum using a framework that integrated formal didactics, interactive online learning, and key clinical components of outpatient nephrology care. Future investigation will evaluate whether early implementation of this curriculum is associated with increased pursuit of nephrology as a career.

SGIM-AMDA-AGS Consensus Best Practice Recommendations for Transitioning Patients' Healthcare from Skilled Nursing Facilities to the Community.

We assembled a cross-cutting team of experts representing primary care physicians (PCPs), home care physicians, physicians who see patients in skilled nursing facilities (SNF physicians), skilled nursing facility medical directors, human factors engineers, transitional care researchers, geriatricians, internists, family practitioners, and three major organizations: AMDA, SGIM, and AGS. This work was sponsored through a grant from the Association of Subspecialty Physicians (ASP). Members of the team mapped the process of discharging patients from a skilled nursing facility into the community and subsequent care of their outpatient PCP. Four areas of process improvement were identified, building on the prior work of the AMDA Transitions of Care Committee and the experiences of the team members. The team identified issues and developed best practices perceived as feasible for SNF physician and PCP practices to accomplish. The goal of these consensus-based recommended best practices is to provide a safe and high-quality transition for patients moving between the care of their SNF physician and PCP.

Year-End Clinic Handoffs: A National Survey of Academic Internal Medicine Programs.

BACKGROUND: While there has been increasing emphasis and innovation nationwide in training residents in inpatient handoffs, very little is known about the practice and preparation for year-end clinic handoffs of residency outpatient continuity practices. Thus, the latter remains an identified, yet nationally unaddressed, patient safety concern. OBJECTIVES: The 2014 annual Association of Program Directors in Internal Medicine (APDIM) survey included seven items for assessing the current year-end clinic handoff practices of internal medicine residency programs throughout the country. DESIGN: Nationwide survey. PARTICIPANTS: All internal medicine program directors registered with APDIM. MAIN MEASURES: Descriptive statistics of programs and tools used to formulate a year-end handoff in the ambulatory setting, methods for evaluating the process, patient safety and quality measures incorporated within the process, and barriers to conducting year-end handoffs. KEY RESULTS: Of the 361 APDIM member programs, 214 (59%) completed the Transitions of Care Year-End Clinic Handoffs section of the survey. Only 34% of respondent programs reported having a year-end ambulatory handoff system, and 4% reported assessing residents for competency in this area. The top three barriers to developing a year-end handoff system were insufficient overlap between graduating and incoming residents, inability to schedule patients with new residents in advance, and time constraints for residents, attendings, and support staff. CONCLUSIONS: Most internal medicine programs do not have a year-end clinic handoff system in place. Greater attention to clinic handoffs and resident assessment of this care transition is needed.

Xenopus: leaping forward in kidney organogenesis.

While kidney donations stagnate, the number of people in need of kidney transplants continues to grow. Although transplanting culture-grown organs is years away, pursuing the engineering of the kidney de novo is a valid means of closing the gap between the supply and demand of kidneys for transplantation. The structural organization of a mouse kidney is similar to that of humans. Therefore, mice have traditionally served as the primary model system for the study of kidney development. The mouse is an ideal model organism for understanding the complexity of the human kidney. Nonetheless, the elaborate structure of the mammalian kidney makes the discovery of new therapies based on de novo engineered kidneys more challenging. In contrast to mammals, amphibians have a kidney that is anatomically less complex and develops faster. Given that analogous genetic networks regulate the development of mammalian and amphibian nephric organs, using embryonic kidneys of Xenopus laevis (African clawed frog) to analyze inductive cell signaling events and morphogenesis has many advantages. Pioneering work that led to the ability to generate kidney organoids from embryonic cells was carried out in Xenopus. In this review, we discuss how Xenopus can be utilized to compliment the work performed in mammalian systems to understand kidney development.

Lung epithelial branching program antagonizes alveolar differentiation.

Mammalian organs, including the lung and kidney, often adopt a branched structure to achieve high efficiency and capacity of their physiological functions. Formation of a functional lung requires two developmental processes: branching morphogenesis, which builds a tree-like tubular network, and alveolar differentiation, which generates specialized epithelial cells for gas exchange. Much progress has been made to understand each of the two processes individually; however, it is not clear whether the two processes are coordinated and how they are deployed at the correct time and location. Here we show that an epithelial branching morphogenesis program antagonizes alveolar differentiation in the mouse lung. We find a negative correlation between branching morphogenesis and alveolar differentiation temporally, spatially, and evolutionarily. Gain-of-function experiments show that hyperactive small GTPase Kras expands the branching program and also suppresses molecular and cellular differentiation of alveolar cells. Loss-of-function experiments show that SRY-box containing gene 9 (Sox9) functions downstream of Fibroblast growth factor (Fgf)/Kras to promote branching and also suppresses premature initiation of alveolar differentiation. We thus propose that lung epithelial progenitors continuously balance between branching morphogenesis and alveolar differentiation, and such a balance is mediated by dual-function regulators, including Kras and Sox9. The resulting temporal delay of differentiation by the branching program may provide new insights to lung immaturity in preterm neonates and the increase in organ complexity during evolution.

Upon Wnt stimulation, Rac1 activation requires Rac1 and Vav2 binding to p120-catenin.

A role for Rac1 GTPase in canonical Wnt signaling has recently been demonstrated, showing that it is required for ß-catenin translocation to the nucleus. In this study, we investigated the mechanism of Rac1 stimulation by Wnt. Upregulation of Rac1 activity by Wnt3a temporally correlated with enhanced p120-catenin binding to Rac1 and Vav2. Vav2 and Rac1 association with p120-catenin was modulated by phosphorylation of this protein, which was stimulated upon serine/threonine phosphorylation by CK1 and inhibited by tyrosine phosphorylation by Src or Fyn. Acting on these two post-translational modifications, Wnt3a induced the release of p120-catenin from E-cadherin, enabled the interaction of p120-catenin with Vav2 and Rac1, and facilitated Rac1 activation by Vav2. Given that p120-catenin depletion disrupts gastrulation in Xenopus, we analyzed p120-catenin mutants for their ability to rescue this phenotype. In contrast to the wild-type protein or other controls, p120-catenin point mutants that were deficient in the release from E-cadherin or in Vav2 or Rac1 binding failed to rescue p120-catenin depletion. Collectively, these results indicate that binding of p120-catenin to Vav2 and Rac1 is required for the activation of this GTPase upon Wnt signaling.

Down's-syndrome-related kinase Dyrk1A modulates the p120-catenin-Kaiso trajectory of the Wnt signaling pathway.

The Wnt pathways contribute to many processes in cancer and development, with ß-catenin being a key canonical component. p120-catenin, which is structurally similar to ß-catenin, regulates the expression of certain Wnt target genes, relieving repression conferred by the POZ- and zinc-finger-domain-containing transcription factor Kaiso. We have identified the kinase Dyrk1A as a component of the p120-catenin-Kaiso trajectory of the Wnt pathway. Using rescue and other approaches in Xenopus laevis embryos and mammalian cells, we found that Dyrk1A positively and selectively modulates p120-catenin protein levels, thus having an impact on p120-catenin and Kaiso (and canonical Wnt) gene targets such as siamois and wnt11. The Dyrk1A gene resides within the Down's syndrome critical region, which is amplified in Down's syndrome. A consensus Dyrk phosphorylation site in p120-catenin was identified, with a mutant mimicking phosphorylation exhibiting the predicted enhanced capacity to promote endogenous Wnt-11 and Siamois expression, and gastrulation defects. In summary, we report the biochemical and functional relationship of Dyrk1A with the p120-catenin-Kaiso signaling trajectory, with a linkage to canonical Wnt target genes. Conceivably, this work might also prove relevant to understanding the contribution of Dyrk1A dosage imbalance in Down's syndrome.

Pronephric tubulogenesis requires Daam1-mediated planar cell polarity signaling.

Canonical ß-catenin-mediated Wnt signaling is essential for the induction of nephron development. Noncanonical Wnt/planar cell polarity (PCP) pathways contribute to processes such as cell polarization and cytoskeletal modulation in several tissues. Although PCP components likely establish the plane of polarization in kidney tubulogenesis, whether PCP effectors directly modulate the actin cytoskeleton in tubulogenesis is unknown. Here, we investigated the roles of Wnt PCP components in cytoskeletal assembly during kidney tubule morphogenesis in Xenopus laevis and zebrafish. We found that during tubulogenesis, the developing pronephric anlagen expresses Daam1 and its interacting Rho-GEF (WGEF), which compose one PCP/noncanonical Wnt pathway branch. Knockdown of Daam1 resulted in reduced expression of late pronephric epithelial markers with no apparent effect upon early markers of patterning and determination. Inhibiting various points in the Daam1 signaling pathway significantly reduced pronephric tubulogenesis. These data indicate that pronephric tubulogenesis requires the Daam1/WGEF/Rho PCP pathway.

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.

UNC-98 links an integrin-associated complex to thick filaments in Caenorhabditis elegans muscle.

Focal adhesions are multiprotein assemblages that link cells to the extracellular matrix. The transmembrane protein, integrin, is a key component of these structures. In vertebrate muscle, focal adhesion-like structures called costameres attach myofibrils at the periphery of muscle cells to the cell membrane. In Caenorhabditis elegans muscle, all the myofibrils are attached to the cell membrane at both dense bodies (Z-disks) and M-lines. Clustered at the base of dense bodies and M-lines, and associated with the cytoplasmic tail of beta-integrin, is a complex of many proteins, including UNC-97 (vertebrate PINCH). Previously, we showed that UNC-97 interacts with UNC-98, a 37-kD protein, containing four C2H2 Zn fingers, that localizes to M-lines. We report that UNC-98 also interacts with the C-terminal portion of a myosin heavy chain. Multiple lines of evidence support a model in which UNC-98 links integrin-associated proteins to myosin in thick filaments at M-lines.

Wnt to build a tube: contributions of Wnt signaling to epithelial tubulogenesis.

Epithelial tubes are crucial to the function of organ systems including the cardiovascular system, pulmonary system, gastrointestinal tract, reproductive organ systems, excretory system, and auditory system. Using a variety of animal model systems, recent studies have substantiated the role of Wnt signaling via the canonical/beta-catenin-mediated trajectory, the non-canonical Wnt trajectories, or both, in forming epithelial tubular tissues. This review focuses on the involvement of the Wnt pathways in the induction, specification, proliferation, and morphogenesis involved in tubulogenesis within tissues including the lungs, kidneys, ears, mammary glands, gut, and heart. The ultimate goal is to describe the developmental processes forming the various tubulogenic organ systems to determine the relationships between these processes.