Aberrant centrosome biogenesis disrupts nephron and collecting duct progenitor growth and fate resulting in fibrocystic kidney disease.

Mutations that disrupt centrosome biogenesis or function cause congenital kidney developmental defects and fibrocystic pathologies. Yet how centrosome dysfunction results in the kidney disease phenotypes remains unknown. Here, we examined the consequences of conditional knockout of the ciliopathy gene Cep120, essential for centrosome duplication, in the nephron and collecting duct progenitor niches of the mouse embryonic kidney. Cep120 loss led to reduced abundance of both cap mesenchyme and ureteric bud populations, due to a combination of delayed mitosis, increased apoptosis and premature differentiation of progenitor cells. These defects resulted in dysplastic kidneys at birth, which rapidly formed cysts, displayed increased interstitial fibrosis and decline in kidney function. RNA sequencing of embryonic and postnatal kidneys from Cep120-null mice identified changes in the pathways essential for development, fibrosis and cystogenesis. Our study defines the cellular and developmental defects caused by centrosome dysfunction during kidney morphogenesis and identifies new therapeutic targets for patients with renal centrosomopathies.

Cep120 is essential for kidney stromal progenitor cell growth and differentiation.

Mutations in genes that disrupt centrosome structure or function can cause congenital kidney developmental defects and lead to fibrocystic pathologies. Yet, it is unclear how defective centrosome biogenesis impacts renal progenitor cell physiology. Here, we examined the consequences of impaired centrosome duplication on kidney stromal progenitor cell growth, differentiation, and fate. Conditional deletion of the ciliopathy gene Cep120, which is essential for centrosome duplication, in the stromal mesenchyme resulted in reduced abundance of interstitial lineages including pericytes, fibroblasts and mesangial cells. These phenotypes were caused by a combination of delayed mitosis, activation of the mitotic surveillance pathway leading to apoptosis, and changes in both Wnt and Hedgehog signaling that are key for differentiation of stromal cells. Cep120 ablation resulted in small hypoplastic kidneys with medullary atrophy and delayed nephron maturation. Finally, Cep120 and centrosome loss in the interstitium sensitized kidneys of adult mice, causing rapid fibrosis after renal injury via enhanced TGF-ß/Smad3-Gli2 signaling. Our study defines the cellular and developmental defects caused by loss of Cep120 and aberrant centrosome biogenesis in the embryonic kidney stroma.

DLG1 functions upstream of SDCCAG3 and IFT20 to control ciliary targeting of polycystin-2.

Polarized vesicular trafficking directs specific receptors and ion channels to cilia, but the underlying mechanisms are poorly understood. Here we describe a role for DLG1, a core component of the Scribble polarity complex, in regulating ciliary protein trafficking in kidney epithelial cells. Conditional knockout of Dlg1 in mouse kidney causes ciliary elongation and cystogenesis, and cell-based proximity labeling proteomics and fluorescence microscopy show alterations in the ciliary proteome upon loss of DLG1. Specifically, the retromer-associated protein SDCCAG3, IFT20, and polycystin-2 (PC2) are reduced in the cilia of DLG1-deficient cells compared to control cells. This phenotype is recapitulated in vivo and rescuable by re-expression of wild-type DLG1, but not a Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)-associated DLG1 variant, p.T489R. Finally, biochemical approaches and Alpha Fold modelling suggest that SDCCAG3 and IFT20 form a complex that associates, at least indirectly, with DLG1. Our work identifies a key role for DLG1 in regulating ciliary protein composition and suggests that ciliary dysfunction of the p.T489R DLG1 variant may contribute to CAKUT.

Bronchiectasis and Bronchiolectasis With Severe Herniating Pattern Associated With STAT1 Gain-of-Function Mutation: Detailed Clinicopathological Findings.

STAT1 gain-of-function (GOF) mutations are associated with a rare autosomal dominant immunodeficiency disorder with main clinical manifestations including chronic mucocutaneous candidiasis (CMC) and bronchiectasis. In addition, these patients show higher incidences of cerebral and extracerebral aneurysm, malignancies and various autoimmune conditions compared to the general population. Although previous publications have reported clinical findings in patients with STAT1 GOF mutation, they did not include histopathologic features. Herein, we describe the first case with detailed histologic findings in the lung of a 5-year-old patient with a de novo STAT1 GOF mutation, who presented with CMC and bronchiectasis. The biopsy showed severe bronchiolectasis with extensive airway dilatation and occasional disruptions. Peribronchiolar inflammation was not always present and evident mainly in areas of airway disruption; inflammation may have not been a main driver of the airway damage in this case. The airway dilatation often showed an interesting herniating pattern, possibly implying a connective tissue etiology. This case also demonstrates the diagnostic utility of whole exome sequencing as STAT1 GOF mutations are not detected by routine workup. The definitive diagnosis will lead to more specific treatments and increased surveillance for serious conditions, such as cerebral aneurysms and malignancies.

The effects of exercise on cardiovascular disease risk factors and cardiovascular physiology in rheumatoid arthritis.

Cardiovascular disease (CVD) morbidity and mortality is highly prevalent in patients with rheumatoid arthritis (RA) with debilitating effects for the individual as well as significant healthcare impact. Current evidence demonstrates that engaging in aerobic and resistance exercise (i.e. structured physical activity) can significantly improve patient-reported and clinical index-assessed outcomes in RA. In addition to this, engagement in exercise programmes improves, in a dose-dependent manner, the risk of developing CVD as well as CVD symptoms and outcomes. The present narrative review uses evidence from systematic reviews and meta-analyses as well as controlled trials, to synthesize the current state-of-the-art on the potential effects of aerobic and resistance exercise on CVD risk factors as well as on cardiac and vascular function and structure in people with RA. Where there is a lack of evidence in RA to explain potential mechanisms, relevant studies from the general population are also discussed and linked to RA.

New pathogenic insights inform therapeutic target development for renal osteodystrophy.

In an ancillary analysis of cross-sectional observational studies of bone health in end-stage kidney disease (ESKD), Evenepoel et al. reported that subjects with autosomal-dominant polycystic kidney disease (ADPKD) had a unique phenotype in their renal osteodystrophy. ADPKD caused resistance to parathyroid hormone (PTH) producing lower turnover states and preservation of cortical bone mineral density. PTH resistance was probably produced by increased osteocyte sclerostin levels, which is regulated by mechanical loading sensed through primary cilia sensory function affected by mutation in PKD1 and PKD2.

Arginine reprogramming in ADPKD results in arginine-dependent cystogenesis.

Research into metabolic reprogramming in cancer has become commonplace, yet this area of research has only recently come of age in nephrology. In light of the parallels between cancer and autosomal dominant polycystic kidney disease (ADPKD), the latter is currently being studied as a metabolic disease. In clear cell renal cell carcinoma (RCC), which is now considered a metabolic disease, we and others have shown derangements in the enzyme arginosuccinate synthase 1 (ASS1), resulting in RCC cells becoming auxotrophic for arginine and leading to a new therapeutic paradigm involving reducing extracellular arginine. Based on our earlier finding that glutamine pathways are reprogrammed in ARPKD, and given the connection between arginine and glutamine synthetic pathways via citrulline, we investigated the possibility of arginine reprogramming in ADPKD. We now show that, in a remarkable parallel to RCC, ASS1 expression is reduced in murine and human ADPKD, and arginine depletion results in a dose-dependent compensatory increase in ASS1 levels as well as decreased cystogenesis in vitro and ex vivo with minimal toxicity to normal cells. Nontargeted metabolomics analysis of mouse kidney cell lines grown in arginine-deficient versus arginine-replete media suggests arginine-dependent alterations in the glutamine and proline pathways. Thus, depletion of this conditionally essential amino acid by dietary or pharmacological means, such as with arginine-degrading enzymes, may be a novel treatment for this disease.

Functional characterization of biallelic RTTN variants identified in an infant with microcephaly, simplified gyral pattern, pontocerebellar hypoplasia, and seizures.

BACKGROUND: Biallelic deleterious variants in RTTN, which encodes rotatin, are associated with primary microcephaly, polymicrogyria, seizures, intellectual disability, and primordial dwarfism in human infants. METHODS AND RESULTS: We performed exome sequencing of an infant with primary microcephaly, pontocerebellar hypoplasia, and intractable seizures and his healthy, unrelated parents. We cultured the infant's fibroblasts to determine primary ciliary phenotype. RESULTS: We identified biallelic variants in RTTN in the affected infant: a novel missense variant and a rare, intronic variant that results in aberrant transcript splicing. Cultured fibroblasts from the infant demonstrated reduced length and number of primary cilia. CONCLUSION: Biallelic variants in RTTN cause primary microcephaly in infants. Functional characterization of primary cilia length and number can be used to determine pathogenicity of RTTN variants.

Anticystogenic activity of a small molecule PAK4 inhibitor may be a novel treatment for autosomal dominant polycystic kidney disease.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common hereditary renal disease with no currently available targeted therapies. Based on the established connection between ß-catenin signaling and renal ciliopathies, and on data from our and other laboratories showing striking similarities of this disease and cancer, we evaluated the use of an orally bioavailable small molecule, KPT-9274 (a dual inhibitor of the protein kinase PAK4 and nicotinamide phosphoribosyl transferase), for treatment of ADPKD. Treatment of PKD-derived cells with this compound not only reduces PAK4 steady-state protein levels and regulates ß-catenin signaling, but also inhibits nicotinamide phosphoribosyl transferase, the rate-limiting enzyme in a key NAD salvage pathway. KPT-9274 can attenuate cellular proliferation and induce apoptosis associated with a decrease in active (phosphorylated) PAK4 and ß-catenin in several Pkd1-null murine cell lines, with a less pronounced effect on the corresponding phenotypically normal cells. Additionally, KPT-9274 shows inhibition of cystogenesis in an ex vivo model of cyclic AMP-induced cystogenesis as well as in the early stage Pkd1flox/flox:Pkhd1-Cre mouse model, the latter showing confirmation of specific anti-proliferative, apoptotic, and on-target effects. NAD biosynthetic attenuation by KPT-9274, while critical for highly proliferative cancer cells, does not appear to be important in the slower growing cystic epithelial cells during cystogenesis. KPT-9274 was not toxic in our ADPKD animal model or in other cancer models. Thus, this small molecule inhibitor could be evaluated in a clinical trial as a viable therapy of ADPKD.

A Complication of Percutaneous Sclerotherapy for Congenital Pulmonary Airway Malformation: Intravascular Injection and Cardiac Necrosis.

INTRODUCTION: A congenital pulmonary airway malformation (CPAM) type III may become large enough to cause hydrops fetalis. In such circumstances, the fetus can be treated with open fetal resection, maternal betamethasone administration, or percutaneous sclerotherapy. CASE REPORT: A 24 week gestation fetus with a CPAM type III was treated by percutaneous sclerotherapy using ethanolamine oleate (EO). The EO inadvertently entered the left atrium and ventricle with subsequent fetal bradycardia and demise. Autopsy revealed myocardial necrosis. CONCLUSION: Percutaneous sclerotherapy has been previously described in the literature for the treatment of microcystic CPAMs with secondary hydrops. This is the first reported case of an adverse event after fetal sclerotherapy.

Avoiding and Managing Intraoperative Complications During Cervical Spine Surgery.

The incidence of intraoperative complications during cervical spine surgery is low; however, if they do occur, intraoperative complications have the potential to cause considerable morbidity and mortality. Spine surgeons should be familiar with methods to minimize intraoperative complications. If they do occur, surgeons must be prepared to immediately treat each potential complication to reduce any associated morbidity.

An Algorithmic Approach to Venous Thromboembolism Prophylaxis in Spine Surgery.

Venous thromboembolic embolism (VTE) is a potentially serious and life-threatening complication in spine surgery. However, VTE incidence and prophylaxis in spine surgery remains controversial. Current recommendations for VTE prophylaxis address "spine surgery" as a single broad category and mainly consider patient factors when determining risk. We performed a literature review to determine the varying VTE and bleeding risks within spine surgery to develop an individualized prophylactic algorithm. Our review suggests that the current guidelines on VTE prophylaxis for spine surgery from NASS and ACCP are suboptimal. Consideration of (1) patient-related VTE risks, (2) procedure-related VTE risks, and (3) the risk of neurological compromise from bleeding complications will more appropriately balance safety and effectiveness when choosing a VTE prophylaxis method. To better individualize VTE prophylaxis, we have developed the VTE Prophylaxis Risk/Benefit Score that considers this currently available best evidence to arrive at a recommendation for the most appropriate form of VTE prophylaxis. This algorithm informs the surgeon to help make a more nuanced and individualized determination of prophylaxis.

A comparison of post-mortem findings in broilers dead-on-farm and broilers dead-on-arrival at the abattoir.

Broiler mortality during transport to abattoirs (dead-on-arrival/DOA) evokes concern due to compromised animal welfare and associated economic losses. The general aim of this study was to characterize pathological lesions associated with mortality in broilers close to slaughter. The specific aim was to investigate whether disease at the end of the growth period may be a predisposing factor for DOA by describing and comparing the pathological findings in broilers dead-on-farm (DOF) in the final days of the production cycle and in broilers DOA from the same flocks. Gross post-mortem examinations were performed on 607 broilers from 32 flocks, either DOF (371) or DOA (236). In DOF broilers, the most common pathological lesions were lung congestion (37.7%), endocarditis (29.4%), and ascites (24.0%), whereas the most common findings in broilers DOA were lung congestion (57.2%) and trauma (24.6%). Lung congestion was more prevalent among DOA broilers compared to DOF broilers (P-value of > 0.001). A possible cause behind the pathological finding lung congestion is sudden death syndrome (SDS). The study indicates that steps in the transportation process per se cause the majority of pathological lesions such as lung congestion and trauma that may have led to the mortalities registered. Pre-existing diseases such as ascites and osteomyelitis may also predispose for DOA. Thus, factors relating to on-farm health, catching, and transportation are all areas of future investigation in order to reduce transport mortalities and to enhance welfare in broilers.

Inhibiting centrosome clustering reduces cystogenesis and improves kidney function in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic disorder accounting for approximately 5% of patients with renal failure, yet therapeutics for the treatment of ADPKD remain limited. ADPKD tissues display abnormalities in the biogenesis of the centrosome, a defect that can cause genome instability, aberrant ciliary signaling, and secretion of pro-inflammatory factors. Cystic cells form excess centrosomes via a process termed centrosome amplification (CA), which causes abnormal multipolar spindle configurations, mitotic catastrophe, and reduced cell viability. However, cells with CA can suppress multipolarity via "centrosome clustering," a key mechanism by which cells circumvent apoptosis. Here, we demonstrate that inhibiting centrosome clustering can counteract the proliferation of renal cystic cells with high incidences of CA. Using ADPKD human cells and mouse models, we show that preventing centrosome clustering with 2 inhibitors, CCB02 and PJ34, blocks cyst initiation and growth in vitro and in vivo. Inhibiting centrosome clustering activates a p53-mediated surveillance mechanism leading to apoptosis, reduced cyst expansion, decreased interstitial fibrosis, and improved kidney function. Transcriptional analysis of kidneys from treated mice identified pro-inflammatory signaling pathways implicated in CA-mediated cystogenesis and fibrosis. Our results demonstrate that centrosome clustering is a cyst-selective target for the improvement of renal morphology and function in ADPKD.

The impact of minimally invasive surgical approaches on surgical-site infections.

We performed a literature review to describe the risk of surgical-site infection (SSI) in minimally invasive surgery (MIS) compared to standard open surgery. Most studies reported decreased SSI rates among patients undergoing MIS compared to open procedures. However, many were observational studies and may have been affected by selection bias. MIS is associated with reduced risk of surgical-site infection compared to standard open surgery and should be considered when feasible.

Multi-omics profiling of mouse polycystic kidney disease progression at a single cell resolution.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and causes significant morbidity, ultimately leading to end-stage kidney disease. PKD pathogenesis is characterized by complex and dynamic alterations in multiple cell types during disease progression, hampering a deeper understanding of disease mechanism and the development of therapeutic approaches. Here, we generate a single nucleus multimodal atlas of an orthologous mouse PKD model at early, mid and late timepoints, consisting of 125,434 single-nucleus transcriptomic and epigenetic multiomes. We catalogue differentially expressed genes and activated epigenetic regions in each cell type during PKD progression, characterizing cell-type-specific responses to Pkd1 deletion. We describe heterogeneous, atypical collecting duct cells as well as proximal tubular cells that constitute cyst epithelia in PKD. The transcriptional regulation of the cyst lining cell marker GPRC5A is conserved between mouse and human PKD cystic epithelia, suggesting shared gene regulatory pathways. Our single nucleus multiomic analysis of mouse PKD provides a foundation to understand the earliest changes molecular deregulation in a mouse model of PKD at a single-cell resolution.

Loss of an extensive ciliary connectome induces proteostasis and cell fate switching in a severe motile ciliopathy.

Motile cilia have essential cellular functions in development, reproduction, and homeostasis. Genetic causes for motile ciliopathies have been identified, but the consequences on cellular functions beyond impaired motility remain unknown. Variants in CCDC39 and CCDC40 cause severe disease not explained by loss of motility. Using human cells with pathological variants in these genes, Chlamydomonas genetics, cryo-electron microscopy, single cell RNA transcriptomics, and proteomics, we identified perturbations in multiple cilia-independent pathways. Absence of the axonemal CCDC39/CCDC40 heterodimer results in loss of a connectome of over 90 proteins. The undocked connectome activates cell quality control pathways, switches multiciliated cell fate, impairs microtubule architecture, and creates a defective periciliary barrier. Both cilia-dependent and independent defects are likely responsible for the disease severity. Our findings provide a foundation for reconsidering the broad cellular impact of pathologic variants in ciliopathies and suggest new directions for therapies.

Characterization of Lumbar Lordosis: Influence of Age, Sex, Vertebral Body Wedging, and L4-S1.

STUDY DESIGN: Retrospective radiographic review. OBJECTIVE: The objectives of the study were to determine the contributions to lumbar lordosis (LL) through both the vertebrae and the intervertebral disc (IVD), and to investigate the relationships between lumbar sagittal spine measurements and age and gender. SUMMARY OF BACKGROUND DATA: A small body of literature exists on the relative contributions of vertebral body and IVD morphology to LL, the effects of L4-S1 on overall LL, and the relationships/correlations between lumbar sagittal spine measurements. METHODS: Patients who met the inclusion criteria were retrospectively evaluated. Measurements included LL, pelvic incidence (PI), and % contributions of vertebral body wedging/IVD wedging/L4-S1 to LL. Patients were separated into groups by age and sex, demographic data were collected, and statistical analysis was completed. RESULTS: LL decreased with age, although PI remained similar. Females demonstrated increased LL and vertebral body wedging % than males. Males demonstrated increased L4-S1% than females. Despite a decrease in LL with age, patients maintained L4-S1% and IVD wedging %. There was a significant negative relationship between PI and IVD wedging, PI and L4-S1%, and LL and L4-S1%. CONCLUSIONS: During aging, the lumbar spine loses LL linearly. This occurs in the IVD and vertebral bodies. Females have increased LL compared with males, because of an increase in vertebral body wedging and IVD/vertebral wedging cranial to L4. In patients with high PI or LL, increased LL occurs from cranial to L4 and from vertebral body wedging.

Ultrastructure expansion microscopy (U-ExM) of mouse and human kidneys for analysis of subcellular structures.

Ultrastructure expansion microscopy (U-ExM) involves the physical magnification of specimens embedded in hydrogels, which allows for super-resolution imaging of subcellular structures using a conventional diffraction-limited microscope. Methods for expansion microscopy exist for several organisms, organs, and cell types, and used to analyze cellular organelles and substructures in nanoscale resolution. Here, we describe a simple step-by-step U-ExM protocol for the expansion, immunostaining, imaging, and analysis of cytoskeletal and organellar structures in kidney tissue. We detail the critical modified steps to optimize isotropic kidney tissue expansion, and preservation of the renal cell structures of interest. We demonstrate the utility of the approach using several markers of renal cell types, centrioles, cilia, the extracellular matrix, and other cytoskeletal elements. Finally, we show that the approach works well on mouse and human kidney samples that were preserved using different fixation and storage conditions. Overall, this protocol provides a simple and cost-effective approach to analyze both pre-clinical and clinical renal samples in high detail, using conventional lab supplies and standard widefield or confocal microscopy.