Comprehensive mapping of sensory and sympathetic innervation of the developing kidney.

The kidney functions as a finely tuned sensor to balance body fluid composition and filter out waste through complex coordinated mechanisms. This versatility requires tight neural control, with innervating efferent nerves playing a crucial role in regulating blood flow, glomerular filtration rate, water and sodium reabsorption, and renin release. In turn sensory afferents provide feedback to the central nervous system for the modulation of cardiovascular function. However, the cells targeted by sensory afferents and the physiological sensing mechanisms remain poorly characterized. Moreover, how the kidney is innervated during development to establish these functions remains elusive. Here, we utilized a combination of light-sheet and confocal microscopy to generate anatomical maps of kidney sensory and sympathetic nerves throughout development and resolve the establishment of functional crosstalk. Our analyses revealed that kidney innervation initiates at embryonic day (E)13.5 as the nerves associate with vascular smooth muscle cells and follow arterial differentiation. By E17.5 axonal projections associate with kidney structures such as glomeruli and tubules and the network continues to expand postnatally. These nerves are synapsin I-positive, highlighting ongoing axonogenesis and the potential for functional crosstalk. We show that sensory and sympathetic nerves innervate the kidney concomitantly and classify the sensory fibers as calcitonin gene related peptide (CGRP)+, substance P+, TRPV1+, and PIEZO2+, establishing the presence of PIEZO2 mechanosensory fibers in the kidney. Using retrograde tracing, we identified the primary dorsal root ganglia, T10-L2, from which PIEZO2+ sensory afferents project to the kidney. Taken together our findings elucidate the temporality of kidney innervation and resolve the identity of kidney sympathetic and sensory nerves.

Netrin 1 directs vascular patterning and maturity in the developing kidney.

The intricate vascular system of the kidneys supports body fluid and organ homeostasis. However, little is known about how vascular architecture is established during kidney development. More specifically, how signals from the kidney influence vessel maturity and patterning remains poorly understood. Netrin 1 (Ntn1) is a secreted ligand that is crucial for vessel and neuronal guidance. Here, we demonstrate that Ntn1 is expressed by Foxd1+ stromal progenitors in the developing mouse kidney and conditional deletion (Foxd1GC/+;Ntn1fl/fl) results in hypoplastic kidneys with extended nephrogenesis. Wholemount 3D analyses additionally revealed the loss of a predictable vascular pattern in Foxd1GC/+;Ntn1fl/fl kidneys. As vascular patterning has been linked to vessel maturity, we investigated arterialization. Quantification of the CD31+ endothelium at E15.5 revealed no differences in metrics such as the number of branches or branch points, whereas the arterial vascular smooth muscle metrics were significantly reduced at both E15.5 and P0. In support of our observed phenotypes, whole kidney RNA-seq revealed disruptions to genes and programs associated with stromal cells, vasculature and differentiating nephrons. Together, our findings highlight the significance of Ntn1 to proper vascularization and kidney development.

Altered binding affinity of SIX1-Q177R correlates with enhanced WNT5A and WNT pathway effector expression in Wilms tumor.

Wilms tumors present as an amalgam of varying proportions of tissues located within the developing kidney, one being the nephrogenic blastema comprising multipotent nephron progenitor cells (NPCs). The recurring missense mutation Q177R in NPC transcription factors SIX1 and SIX2 is most correlated with tumors of blastemal histology and is significantly associated with relapse. Yet, the transcriptional regulatory consequences of SIX1/2-Q177R that might promote tumor progression and recurrence have not been investigated extensively. Utilizing multiple Wilms tumor transcriptomic datasets, we identified upregulation of the gene encoding non-canonical WNT ligand WNT5A in addition to other WNT pathway effectors in SIX1/2-Q177R mutant tumors. SIX1 ChIP-seq datasets from Wilms tumors revealed shared binding sites for SIX1/SIX1-Q177R within a promoter of WNT5A and at putative distal cis-regulatory elements (CREs). We demonstrate colocalization of SIX1 and WNT5A in Wilms tumor tissue and utilize in vitro assays that support SIX1 and SIX1-Q177R activation of expression from the WNT5A CREs, as well as enhanced binding affinity within the WNT5A promoter that may promote the differential expression of WNT5A and other WNT pathway effectors associated with SIX1-Q177R tumors.

Mapping of the podocin proximity-dependent proteome reveals novel components of the kidney podocyte foot process.

Introduction: The unique architecture of glomerular podocytes is integral to kidney filtration. Interdigitating foot processes extend from the podocyte cell body, wrap around fenestrated capillaries, and form specialized junctional complexes termed slit diaphragms to create a molecular sieve. However, the full complement of proteins which maintain foot process integrity, and how this localized proteome changes with disease, remain to be elucidated. Methods: Proximity-dependent biotin identification (BioID) enables the identification of spatially localized proteomes. To this end, we developed a novel in vivo BioID knock-in mouse model. We utilized the slit diaphragm protein podocin (Nphs2) to create a podocin-BioID fusion. Podocin-BioID localizes to the slit diaphragm, and biotin injection leads to podocyte-specific protein biotinylation. We isolated the biotinylated proteins and performed mass spectrometry to identify proximal interactors. Results and Discussion: Gene ontology analysis of 54 proteins specifically enriched in our podocin-BioID sample revealed 'cell junctions,' 'actin binding,' and 'cytoskeleton organization' as top terms. Known foot process components were identified, and we further uncovered two novel proteins: the tricellular junctional protein Ildr2 and the CDC42 and N-WASP interactor Fnbp1l. We confirmed that Ildr2 and Fnbp1l are expressed by podocytes and partially colocalize with podocin. Finally, we investigated how this proteome changes with age and uncovered a significant increase in Ildr2. This was confirmed by immunofluorescence on human kidney samples and suggests altered junctional composition may preserve podocyte integrity. Together, these assays have led to new insights into podocyte biology and support the efficacy of utilizing BioID in vivo to interrogate spatially localized proteomes in health, aging, and disease.

Netrin-1 directs vascular patterning and maturity in the developing kidney.

Blood filtering by the kidney requires the establishment of an intricate vascular system that works to support body fluid and organ homeostasis. Despite these critical roles, little is known about how vascular architecture is established during kidney development. More specifically, how signals from the kidney influence vessel maturity and patterning remains poorly understood. Netrin-1 (Ntn1) is a secreted ligand critical for vessel and neuronal guidance. Here, we demonstrate that Ntn1 is expressed by stromal progenitors in the developing kidney, and conditional deletion of Ntn1 from Foxd1+ stromal progenitors (Foxd1GC/+;Ntn1fl/fl) results in hypoplastic kidneys that display extended nephrogenesis. Despite expression of the netrin-1 receptor Unc5c in the adjacent nephron progenitor niche, Unc5c knockout kidneys develop normally. The netrin-1 receptor Unc5b is expressed by embryonic kidney endothelium and therefore we interrogated the vascular networks of Foxd1GC/+;Ntn1fl/fl kidneys. Wholemount, 3D analyses revealed the loss of a predictable vascular pattern in mutant kidneys. As vascular patterning has been linked to vessel maturity, we investigated arterialization in these mutants. Quantification of the CD31+ endothelium at E15.5 revealed no differences in metrics such as the number of branches or branch points, whereas the arterial vascular smooth muscle metrics were significantly reduced at both E15.5 and P0. In support of these results, whole kidney RNA-seq showed upregulation of angiogenic programs and downregulation of muscle-related programs which included smooth muscle-associated genes. Together, our findings highlight the significance of netrin-1 to proper vascularization and kidney development.

Sex differences in resilience to ferroptosis underlie sexual dimorphism in kidney injury and repair.

In both humans and mice, repair of acute kidney injury is worse in males than in females. Here, we provide evidence that this sexual dimorphism results from sex differences in ferroptosis, an iron-dependent, lipid-peroxidation-driven regulated cell death. Using genetic and single-cell transcriptomic approaches in mice, we report that female sex confers striking protection against ferroptosis, which was experimentally induced in proximal tubular (PT) cells by deleting glutathione peroxidase 4 (Gpx4). Single-cell transcriptomic analyses further identify the NFE2-related factor 2 (NRF2) antioxidant protective pathway as a female resilience mechanism against ferroptosis. Genetic inhibition and pharmacological activation studies show that NRF2 controls PT cell fate and plasticity by regulating ferroptosis. Importantly, pharmacological NRF2 activation protects male PT cells from ferroptosis and improves cellular plasticity as in females. Our data highlight NRF2 as a potential therapeutic target to prevent failed renal repair after acute kidney injury in both sexes by modulating cellular plasticity.

Innervation in organogenesis.

Proper innervation of peripheral organs helps to maintain physiological homeostasis and elicit responses to external stimuli. Disruptions to normal function can result in pathophysiological consequences. The establishment of connections and communication between the central nervous system and the peripheral organs is accomplished through the peripheral nervous system. Neuronal connections with target tissues arise from ganglia partitioned throughout the body. Organ innervation is initiated during development with stimuli being conducted through several types of neurons including sympathetic, parasympathetic, and sensory. While the physiological modulation of mature organs by these nerves is largely understood, their role in mammalian development is only beginning to be uncovered. Interactions with cells in target tissues can affect the development and eventual function of several organs, highlighting their significance. This chapter will cover the origin of peripheral neurons, factors mediating organ innervation, and the composition and function of organ-specific nerves during development. This emerging field aims to identify the functional contribution of innervation to development which will inform future investigations of normal and abnormal mammalian organogenesis, as well as contribute to regenerative and organ replacement efforts where nerve-derived signals may have significant implications for the advancement of such studies.

Ferroptotic stress promotes the accumulation of pro-inflammatory proximal tubular cells in maladaptive renal repair.

Overwhelming lipid peroxidation induces ferroptotic stress and ferroptosis, a non-apoptotic form of regulated cell death that has been implicated in maladaptive renal repair in mice and humans. Using single-cell transcriptomic and mouse genetic approaches, we show that proximal tubular (PT) cells develop a molecularly distinct, pro-inflammatory state following injury. While these inflammatory PT cells transiently appear after mild injury and return to their original state without inducing fibrosis, after severe injury they accumulate and contribute to persistent inflammation. This transient inflammatory PT state significantly downregulates glutathione metabolism genes, making the cells vulnerable to ferroptotic stress. Genetic induction of high ferroptotic stress in these cells after mild injury leads to the accumulation of the inflammatory PT cells, enhancing inflammation and fibrosis. Our study broadens the roles of ferroptotic stress from being a trigger of regulated cell death to include the promotion and accumulation of proinflammatory cells that underlie maladaptive repair.

Injection of Evans blue dye to fluorescently label and image intact vasculature.

Blood vessels perform critical functions in both health and disease. Understanding how vessels form, pattern and respond to damage is essential. However, labeling and imaging the vasculature to ascertain these properties can be difficult and time-consuming. Here, the authors present a novel methodology for rapidly and efficiently labeling whole vascular networks in vivo by exploiting the fluorescent properties of Evans blue. By combining the labeling with fluorescence microscopy, this method enables visualization of whole tissue vasculature for a fraction of the time and cost compared with traditional methods.

Implementing a Goal-Directed Therapy Protocol for Fluid Resuscitation in the Cardiovascular Intensive Care Unit.

BACKGROUND: Balancing fluid administration and titration of vasoactive medications is critical to preventing postoperative complications in cardiac surgical patients. OBJECTIVE: To evaluate the impact of implementing a goal-directed therapy protocol in the cardiovascular intensive care unit on total intravenous fluids administered on the day of surgery, rates of acute kidney injury, and hospital length of stay. METHODS: A fluid resuscitation protocol using dynamic assessment of fluid responsiveness with stroke volume index was developed, and nurses were prepared for its implementation using simulation training. RESULTS: After implementation of the new protocol, the total amount of intravenous fluids administered on the day of surgery was significantly reduced (P = .003). There were no significant changes in hospital length of stay (P = .83) or rates of acute kidney injury (P = .86). There were significant increases in nurses' knowledge of (P < .001) and confidence in (P < .001) fluid resuscitation and titration of vasoactive medications after simulation training. CONCLUSIONS: Use of a fluid resuscitation protocol resulted in a reduction in the amount of intravenous fluids administered on the day of surgery. The simulation training increased nurses' knowledge of and confidence in fluid resuscitation and titration of vasoactive medications.

Content and Performance of the MiniMUGA Genotyping Array: A New Tool To Improve Rigor and Reproducibility in Mouse Research.

The laboratory mouse is the most widely used animal model for biomedical research, due in part to its well-annotated genome, wealth of genetic resources, and the ability to precisely manipulate its genome. Despite the importance of genetics for mouse research, genetic quality control (QC) is not standardized, in part due to the lack of cost-effective, informative, and robust platforms. Genotyping arrays are standard tools for mouse research and remain an attractive alternative even in the era of high-throughput whole-genome sequencing. Here, we describe the content and performance of a new iteration of the Mouse Universal Genotyping Array (MUGA), MiniMUGA, an array-based genetic QC platform with over 11,000 probes. In addition to robust discrimination between most classical and wild-derived laboratory strains, MiniMUGA was designed to contain features not available in other platforms: (1) chromosomal sex determination, (2) discrimination between substrains from multiple commercial vendors, (3) diagnostic SNPs for popular laboratory strains, (4) detection of constructs used in genetically engineered mice, and (5) an easy-to-interpret report summarizing these results. In-depth annotation of all probes should facilitate custom analyses by individual researchers. To determine the performance of MiniMUGA, we genotyped 6899 samples from a wide variety of genetic backgrounds. The performance of MiniMUGA compares favorably with three previous iterations of the MUGA family of arrays, both in discrimination capabilities and robustness. We have generated publicly available consensus genotypes for 241 inbred strains including classical, wild-derived, and recombinant inbred lines. Here, we also report the detection of a substantial number of XO and XXY individuals across a variety of sample types, new markers that expand the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. We provide preliminary evidence that the array can be used to identify both partial sex chromosome duplication and mosaicism, and that diagnostic SNPs can be used to determine how long inbred mice have been bred independently from the relevant main stock. We conclude that MiniMUGA is a valuable platform for genetic QC, and an important new tool to increase the rigor and reproducibility of mouse research.

The vaping epidemic: Calling nurses to action.

Youth e-cigarette use was declared a national epidemic in 2018. This article discusses e-cigarette- or vaping-associated lung injury (EVALI) and highlights the unique role nurses can have as advocates, patient educators, and champions of health promotion and disease prevention for their patients and families.

Enhancing Family-Centered Care in Cardiothoracic Surgery.

The psychological impact of critical illness is far reaching, affecting patients and their loved ones. Family members face a multitude of stressors, ranging from concerns about death or permanent disability to stress over health care costs and lost wages. Patients are at risk for developing post-intensive care syndrome. Professional groups and patient safety organizations have crafted family-centered care (FCC) models that support hospitalized patients and their families. There is a paucity of data on use of FCC in cardiothoracic intensive care units. This article discusses FCC models and why they are beneficial to the needs of families of postoperative cardiothoracic surgery patients.

Implementing a Weaning Protocol for Cardiac Surgery Patients Using Simulation: A Quality Improvement Project.

BACKGROUND: Mechanical ventilation is the standard of care after cardiac surgery, but it imposes physiologic and psychological stress on patients. The Society of Thoracic Surgery recommends 6 hours as the goal for extubation, but 60% of our patients were not meeting this metric. OBJECTIVES: The objectives of this project were to decrease cardiac surgery patients' ventilation hours and intensive care unit length of stay using a ventilator weaning protocol. METHODS: An evidence-based ventilator weaning protocol was developed, and nurses were prepared for its implementation using a simulation education program. RESULTS: Ventilator hours were reduced from 7.74 to 6.27 (t = 2.5, P = .012). The percentage of patients extubated in 6 hours increased from 40% to 63.5% (χ = 7.757, P = .005). There was no statistically significant decrease in cardiovascular intensive care unit length of stay (17.15 to 15.99, t = 0.619, P = .537). Nurses' scores on a knowledge test increased significantly from pre (6.11) to post (7.79) (t = -5.04, P < .001). Their perception of confidence increased in weaning from pre (median, 4; IQR, 4,4) to post (median, 4; interquartile range [IQR], 4,5), z = -2.71, P = .007, and also in using the protocol from pre (median, 4; IQR, 3,4) to post (median, 4; IQR, 4,5) (z = -3.17, P = .002). DISCUSSION: Using a nurse-led ventilator weaning protocol resulted in decreased ventilator hours for patients and increased knowledge and confidence for nurses.

Anticoagulant Medications for the Prevention and Treatment of Thromboembolism.

Venous thromboembolism is a preventable medical condition associated with significant morbidity and mortality. It can lead to deep vein thrombosis, pulmonary embolism, and stroke. Thrombi develop when intravascular conditions promote activation of the coagulation system or when there is an imbalance between endogenous anticoagulants and procoagulants. Such conditions include vascular injury, inflammation, venous stasis, and hypercoagulable states. Anticoagulant medications are indicated for the prevention and treatment of venous thromboembolism. They exert their effect on clotting factors to prevent the formation of thrombi or the propagation of an existing clot. Historically, anticoagulants were limited to heparins and vitamin K antagonists. Over the past 15 years, however, several new anticoagulant medications have been introduced. This article describes commonly prescribed and newer anticoagulants available to health care professionals, including their mechanism of action, therapeutic use, unique characteristics, and available reversal agents in the event of life-threatening bleeding.

Nephron progenitor cell commitment: Striking the right balance.

The filtering component of the kidney, the nephron, arises from a single progenitor population. These nephron progenitor cells (NPCs) both self-renew and differentiate throughout the course of kidney development ensuring sufficient nephron endowment. An appropriate balance of these processes must be struck as deficiencies in nephron numbers are associated with hypertension and kidney disease. This review will discuss the mechanisms and molecules supporting NPC maintenance and differentiation. A focus on recent work will highlight new molecular insights into NPC regulation and their dynamic behavior in both space and time.

Wnt11 directs nephron progenitor polarity and motile behavior ultimately determining nephron endowment.

A normal endowment of nephrons in the mammalian kidney requires a balance of nephron progenitor self-renewal and differentiation throughout development. Here, we provide evidence for a novel action of ureteric branch tip-derived Wnt11 in progenitor cell organization and interactions within the nephrogenic niche, ultimately determining nephron endowment. In Wnt11 mutants, nephron progenitors dispersed from their restricted niche, intermixing with interstitial progenitors. Nephron progenitor differentiation was accelerated, kidneys were significantly smaller, and the nephron progenitor pool was prematurely exhausted, halving the final nephron count. Interestingly, RNA-seq revealed no significant differences in gene expression. Live imaging of nephron progenitors showed that in the absence of Wnt11 they lose stable attachments to the ureteric branch tips, continuously detaching and reattaching. Further, the polarized distribution of several markers within nephron progenitors is disrupted. Together these data highlight the importance of Wnt11 signaling in directing nephron progenitor behavior which determines a normal nephrogenic program.

Transcriptional regulatory control of mammalian nephron progenitors revealed by multi-factor cistromic analysis and genetic studies.

Nephron progenitor number determines nephron endowment; a reduced nephron count is linked to the onset of kidney disease. Several transcriptional regulators including Six2, Wt1, Osr1, Sall1, Eya1, Pax2, and Hox11 paralogues are required for specification and/or maintenance of nephron progenitors. However, little is known about the regulatory intersection of these players. Here, we have mapped nephron progenitor-specific transcriptional networks of Six2, Hoxd11, Osr1, and Wt1. We identified 373 multi-factor associated 'regulatory hotspots' around genes closely associated with progenitor programs. To examine their functional significance, we deleted 'hotspot' enhancer elements for Six2 and Wnt4. Removal of the distal enhancer for Six2 leads to a ~40% reduction in Six2 expression. When combined with a Six2 null allele, progeny display a premature depletion of nephron progenitors. Loss of the Wnt4 enhancer led to a significant reduction of Wnt4 expression in renal vesicles and a mildly hypoplastic kidney, a phenotype also enhanced in combination with a Wnt4 null mutation. To explore the regulatory landscape that supports proper target gene expression, we performed CTCF ChIP-seq to identify insulator-boundary regions. One such putative boundary lies between the Six2 and Six3 loci. Evidence for the functional significance of this boundary was obtained by deep sequencing of the radiation-induced Brachyrrhine (Br) mutant allele. We identified an inversion of the Six2/Six3 locus around the CTCF-bound boundary, removing Six2 from its distal enhancer regulation, but placed next to Six3 enhancer elements which support ectopic Six2 expression in the lens where Six3 is normally expressed. Six3 is now predicted to fall under control of the Six2 distal enhancer. Consistent with this view, we observed ectopic Six3 in nephron progenitors. 4C-seq supports the model for Six2 distal enhancer interactions in wild-type and Br/+ mouse kidneys. Together, these data expand our view of the regulatory genome and regulatory landscape underpinning mammalian nephrogenesis.