Implementation and Feasibility of Clinical Genome Sequencing Embedded Into the Outpatient Nephrology Care for Patients With Proteinuric Kidney Disease.

Introduction: The diagnosis and management of proteinuric kidney diseases such as focal segmental glomerulosclerosis (FSGS) are challenging. Genetics holds the promise to improve clinical decision making for these diseases; however, it is often performed too late to enable timely clinical action and it is not implemented within routine outpatient nephrology visits. Methods: We sought to test the implementation and feasibility of clinical rapid genome sequencing (GS) in guiding decision making in patients with proteinuric kidney disease in real-time and embedded in the outpatient nephrology setting. Results: We enrolled 10 children or young adults with biopsy-proven FSGS (9 cases) or minimal change disease (1 case). The mean age at enrollment was 16.2 years (range 2-30). The workflow did not require referral to external genetics clinics but was conducted entirely during the nephrology standard-of-care appointments. The total turn-around-time from enrollment to return-of-results and clinical decision averaged 21.8 days (12.4 for GS), which is well within a time frame that allows clinically relevant treatment decisions. A monogenic or APOL1-related form of kidney disease was diagnosed in 5 of 10 patients. The genetic findings resulted in a rectified diagnosis in 6 patients. Both positive and negative GS findings determined a change in pharmacological treatment. In 3 patients, the results were instrumental for transplant evaluation, donor selection, and the immunosuppressive treatment. All patients and families received genetic counseling. Conclusion: Clinical GS is feasible and can be implemented in real-time in the outpatient care to help guiding clinical management. Additional studies are needed to confirm the cost-effectiveness and broader utility of clinical GS across the phenotypic and demographic spectrum of kidney diseases.

The effect of genetic education on the referral of patients to genetic evaluation: Findings from a national survey of nephrologists.

PURPOSE: The success of genomic medicine hinges on the implementation of genetic knowledge in clinical settings. In novel subspecialties, it requires that clinicians refer patients to genetic evaluation or testing, however referral is likely to be affected by genetic knowledge. METHODS: An online survey was administered to self-identified nephrologists working in the United States. Nephrologists' demographic characteristics, genetic education, confidence in clinical genetics, genetic knowledge, and referral rates of patients to genetic evaluation were collected. RESULTS: In total, 201 nephrologists completed the survey. All reported treating patients with genetic forms of kidney disease, and 37% had referred <5 patients to genetic evaluation. A third had limited basic genetic knowledge. Most nephrologists (85%) reported concerns regarding future health insurance eligibility as a barrier to referral to genetic testing. Most adult nephrologists reported insufficient genetic education during residency (65%) and fellowship training (52%). Lower rating of genetic education and lower knowledge in recognizing signs of genetic kidney diseases were significantly associated with lower number of patients referred to the genetic evaluation (P < .001). Most nephrologists reported that improving their genetic knowledge is important for them (>55%). CONCLUSIONS: There is a need to enhance nephrologists' genetic education to increase genetic testing use in nephrology.

Low nephron endowment increases susceptibility to renal stress and chronic kidney disease.

Preterm birth results in low nephron endowment and increased risk of acute kidney injury (AKI) and chronic kidney disease (CKD). To understand the pathogenesis of AKI and CKD in preterm humans, we generated potentially novel mouse models with a 30%-70% reduction in nephron number by inhibiting or deleting Ret tyrosine kinase in the developing ureteric bud. These mice developed glomerular and tubular hypertrophy, followed by the transition to CKD, recapitulating the renal pathological changes seen in humans born preterm. We injected neonatal mice with gentamicin, a ubiquitous nephrotoxic exposure in preterm infants, and detected more severe proximal tubular injury in mice with low nephron number compared with controls with normal nephron number. Mice with low nephron number had reduced proliferative repair with more rapid development of CKD. Furthermore, mice had more profound inflammation with highly elevated levels of MCP-1 and CXCL10, produced in part by damaged proximal tubules. Our study directly links low nephron endowment with postnatal renal hypertrophy, which in this model is maladaptive and results in CKD. Underdeveloped kidneys are more susceptible to gentamicin-induced AKI, suggesting that AKI in the setting of low nephron number is more severe and further increases the risk of CKD in this vulnerable population.

Snapshots of nascent RNA reveal cell- and stimulus-specific responses to acute kidney injury.

The current strategy to detect acute injury of kidney tubular cells relies on changes in serum levels of creatinine. Yet serum creatinine (sCr) is a marker of both functional and pathological processes and does not adequately assay tubular injury. In addition, sCr may require days to reach diagnostic thresholds, yet tubular cells respond with programs of damage and repair within minutes or hours. To detect acute responses to clinically relevant stimuli, we created mice expressing Rosa26-floxed-stop uracil phosphoribosyltransferase (Uprt) and inoculated 4-thiouracil (4-TU) to tag nascent RNA at selected time points. Cre-driven 4-TU-tagged RNA was isolated from intact kidneys and demonstrated that volume depletion and ischemia induced different genetic programs in collecting ducts and intercalated cells. Even lineage-related cell types expressed different genes in response to the 2 stressors. TU tagging also demonstrated the transient nature of the responses. Because we placed Uprt in the ubiquitously active Rosa26 locus, nascent RNAs from many cell types can be tagged in vivo and their roles interrogated under various conditions. In short, 4-TU labeling identifies stimulus-specific, cell-specific, and time-dependent acute responses that are otherwise difficult to detect with other technologies and are entirely obscured when sCr is the sole metric of kidney damage.

Copy Number Variant Analysis and Genome-wide Association Study Identify Loci with Large Effect for Vesicoureteral Reflux.

BACKGROUND: Vesicoureteral reflux (VUR) is a common, familial genitourinary disorder, and a major cause of pediatric urinary tract infection (UTI) and kidney failure. The genetic basis of VUR is not well understood. METHODS: A diagnostic analysis sought rare, pathogenic copy number variant (CNV) disorders among 1737 patients with VUR. A GWAS was performed in 1395 patients and 5366 controls, of European ancestry. RESULTS: Altogether, 3% of VUR patients harbored an undiagnosed rare CNV disorder, such as the 1q21.1, 16p11.2, 22q11.21, and triple X syndromes ((OR, 3.12; 95% CI, 2.10 to 4.54; P=6.35×10-8) The GWAS identified three study-wide significant and five suggestive loci with large effects (ORs, 1.41-6.9), containing canonical developmental genes expressed in the developing urinary tract (WDPCP, OTX1, BMP5, VANGL1, and WNT5A). In particular, 3.3% of VUR patients were homozygous for an intronic variant in WDPCP (rs13013890; OR, 3.65; 95% CI, 2.39 to 5.56; P=1.86×10-9). This locus was associated with multiple genitourinary phenotypes in the UK Biobank and eMERGE studies. Analysis of Wnt5a mutant mice confirmed the role of Wnt5a signaling in bladder and ureteric morphogenesis. CONCLUSIONS: These data demonstrate the genetic heterogeneity of VUR. Altogether, 6% of patients with VUR harbored a rare CNV or a common variant genotype conferring an OR >3. Identification of these genetic risk factors has multiple implications for clinical care and for analysis of outcomes in VUR.

AKI in COVID-19-Associated Multisystem Inflammatory Syndrome in Children (MIS-C).

Background: Multisystem inflammatory syndrome in children (MIS-C) is a recently identified entity in association with COVID-19. AKI has been widely reported in patients with primary COVID-19 infection. However, there is a paucity of literature regarding renal injury in MIS-C. We aim to characterize AKI in MIS-C in this cohort identified at a major children's hospital in New York City during the COVID-19 pandemic. Methods: We conducted a retrospective cohort study of children 0-20 years old admitted to Morgan Stanley Children's Hospital (MSCH) between April 18th and September 23rd, 2020. Patients were included if they met criteria for MIS-C on the basis of CDC guidelines. All patients were evaluated for the presence of AKI, and AKI was staged according to KDIGO criteria. Results: Of the 57 children who met inclusion criteria, 46% (26 of 57) were found to have AKI. The majority of patients (58%; 15 of 26) were classified as KDIGO stage 1. AKI was present upon admission in 70% of those identified. All patients had resolution of AKI at discharge, with 61% achieving recovery by day 2. One patient required dialysis. When compared with those without renal injury, the AKI cohort was older (P<0.001) and had higher median peak values of CRP (P<0.001), IL-6 (P=0.02), ferritin (P<0.001), and procalcitonin (P=0.02). More patients with AKI had left ventricular systolic dysfunction (P<0.001) and lymphopenia (P=0.01) when compared with those without AKI. No differences in body mass index or sex were found. Conclusions: Although children with MIS-C may develop AKI, our study suggests that most experience mild disease, swift resolution, and promising outcome. Older age, increased inflammation, and left ventricular systolic dysfunction may be risk factors. Our study highlights the substantial differences in epidemiology and outcomes between AKI associated with pediatric MIS-C versus primary COVID-19 infection.

Molecular regulation and function of FoxO3 in chronic kidney disease.

PURPOSE OF REVIEW: FOXOs are transcription factors that regulate downstream target genes to counteract to cell stress. Here we review the function and regulation of FOXO transcription factors, the mechanism of FOXO3 activation in the kidney, and the role of FOXO3 in delaying the development of chronic kidney disease (CKD). RECENT FINDINGS: Progressive renal hypoxia from vascular dropout and metabolic perturbation is a pathogenic factor for the initiation and development of CKD. Hypoxia and low levels of α-ketoglutarate generated from the TCA cycle inhibit prolyl hydroxylase domain (PHD)-mediated prolyl hydroxylation of FoxO3, thus reducing FoxO3 protein degradation via the ubiquitin proteasomal pathway, similar to HIF stabilization under hypoxic conditions. FoxO3 accumulation and nuclear translocation activate two key cellular defense mechanisms, autophagy and antioxidative response in renal tubular cells, to reduce cell injury and promote cell survival. FoxO3 directly activates the expression of Atg proteins, which replenishes core components of the autophagic machinery to allow sustained autophagy in the chronically hypoxic kidney. FoxO3 protects mitochondria by stimulating the expression of superoxide dismutase 2 (SOD2), as tubular deletion of FoxO3 in mice results in reduced SOD2 levels and profound mitochondrial damage. SUMMARY: Knowledge gained from animal studies may help understand the function of stress responsive transcription factors that could be targeted to prevent or treat CKD.

Restraining Lysosomal Activity Preserves Hematopoietic Stem Cell Quiescence and Potency.

Quiescence is a fundamental property that maintains hematopoietic stem cell (HSC) potency throughout life. Quiescent HSCs are thought to rely on glycolysis for their energy, but the overall metabolic properties of HSCs remain elusive. Using combined approaches, including single-cell RNA sequencing (RNA-seq), we show that mitochondrial membrane potential (MMP) distinguishes quiescent from cycling-primed HSCs. We found that primed, but not quiescent, HSCs relied readily on glycolysis. Notably, in vivo inhibition of glycolysis enhanced the competitive repopulation ability of primed HSCs. We further show that HSC quiescence is maintained by an abundance of large lysosomes. Repression of lysosomal activation in HSCs led to further enlargement of lysosomes while suppressing glucose uptake. This also induced increased lysosomal sequestration of mitochondria and enhanced the competitive repopulation ability of primed HSCs by over 90-fold in vivo. These findings show that restraining lysosomal activity preserves HSC quiescence and potency and may be therapeutically relevant.

FoxO3 activation in hypoxic tubules prevents chronic kidney disease.

Acute kidney injury (AKI) can lead to chronic kidney disease (CKD) if injury is severe and/or repair is incomplete. However, the pathogenesis of CKD following renal ischemic injury is not fully understood. Capillary rarefaction and tubular hypoxia are common findings during the AKI to CKD transition. We investigated the tubular stress response to hypoxia and demonstrated that a stress responsive transcription factor, FoxO3, was regulated by prolyl hydroxylase. Hypoxia inhibited FoxO3 prolyl hydroxylation and FoxO3 degradation, thus leading to FoxO3 accumulation and activation in tubular cells. Hypoxia-activated Hif-1α contributed to FoxO3 activation and functioned to protect kidneys, as tubular deletion of Hif-1α decreased hypoxia-induced FoxO3 activation, and resulted in more severe tubular injury and interstitial fibrosis following ischemic injury. Strikingly, tubular deletion of FoxO3 during the AKI to CKD transition aggravated renal structural and functional damage leading to a more profound CKD phenotype. We showed that tubular deletion of FoxO3 resulted in decreased autophagic response and increased oxidative injury, which may explain renal protection by FoxO3. Our study indicates that in the hypoxic kidney, stress responsive transcription factors can be activated for adaptions to counteract hypoxic insults, thus attenuating CKD development.

Patent ductus arteriosus is associated with acute kidney injury in the preterm infant.

BACKGROUND: This study aimed to test the hypothesis that a patent ductus arteriosus (PDA) is independently associated with acute kidney injury (AKI) in neonates ≤ 28 weeks gestation. METHODS: Preterm infants with echocardiographic diagnosis of moderate-large PDA at age ≤ 30 days were studied retrospectively. AKI, the primary outcome, was defined and staged according to serum creatinine using Kidney Disease Improving Global Outcomes (KDIGO) neonatal criteria. Its association with the timing and duration of PDA, non-steroidal anti-inflammatory drugs (NSAIDs) and other nephrotoxic exposures, gestational age, and other covariates was evaluated using mixed-effects logistic regression models. RESULTS: Acute Kidney Injury occurred in 49% (101/206) of infants. Moderate-to-large PDA was associated with any-stage AKI (OR 5.31, 95% CI 3.75 to 7.53), stage 1 (mild) AKI (OR 4.86, 95% CI 3.12 to 7.56), and stages 2-3 (severe) AKI (OR 10.9, 95% CI 5.70 to 20.8). NSAID treatment added additional risk for mild AKI (OR 2.45, 95% CI 1.61 to 3.71). Severe AKI was less likely when NSAID treatment was effective (OR 0.45, 95% CI 0.21 to 0.97) but not when ineffective (OR 1.63, 95% CI 0.76 to 3.50). CONCLUSIONS: Moderate-to-large PDA was strongly associated with all stages of AKI in preterm infants ≤ 28 weeks of gestational age. Effective NSAID treatment decreased the risk of severe but not mild AKI. These differential effects reflect the balance between the renal benefits of PDA closure and the risk of NSAID toxicity.

Efficiency and performance tests of the sorptive building materials that reduce indoor formaldehyde concentrations.

The adsorption of volatile organic compounds by building materials reduces the pollutant concentrations in indoor air. We collected three interior building materials with adsorption potentials-latex paint, micro-carbonized plywood, and moisture-buffering siding-used the sorptive building materials test (SBMT) to determine how much they reduced indoor formaldehyde (HCHO) concentrations, and then assessed the consequent reduction in human cancer risk from HCHO inhalation. Adsorption of HCHO by building materials significantly improved the effective ventilation efficiency. For example, the equivalent ventilation rate for Celite siding-used for humidity control-was 1.44 m3/(m2·h) at 25°C, 50% relative humidity (RH); the loading factor (L) was 0.4 m2/m3, and the HCHO concentration was 0.2 ppm; this effect is equivalent to a higher ventilation rate of approximately 0.6 air changes per hour in a typical Taiwanese dwelling. There was also a substantial reduction of risk in Case MCP-2 (Cin,te: 245 µg/m3, 30°C, 50% RH): males: down 5.73 × 10-4; females: down 4.84 × 10-4). The selection of adsorptive building materials for interior surfaces, therefore, significantly reduces human inhalation of HCHO. Our findings should encourage developing and using innovative building materials that help improve indoor air quality and thus provide building occupants with healthier working and living environments.

Sandwich Technique for Endovascular Repair of Acute Type A Aortic Dissection.

OVERVIEW: To describe a new endovascular procedure for acute type A aortic dissection (TAAD) repair. METHODS: Between 2013 and 2016, 12 patients (average age 54±9.6 years; 10 men) with acute TAAD (mean EURO score 11.4%±3.2%, range 5-17) and unfit for surgery underwent thoracic endovascular aortic repair (TEVAR) with 2 periscope grafts to preserve blood supply to supra-aortic branches plus bypass grafting as needed. If the ascending aorta was dilated to >40 mm, sternotomy was performed to wrap the ascending aorta and reduce its diameter to accommodate the aortic stent-grafts. RESULTS: All patients were successfully treated. Seven patients required bypass grafting, and most of the patients had periscope grafts to the innominate/right common carotid artery and left common carotid artery; only 3 patients had the left subclavian artery preserved. All patients exhibited good hemodynamics and normal pressures after the procedure. The mean procedure time and blood loss were 4.5±1.0 hours and 217±111.5 mL, respectively. Two patients treated emergently died shortly after surgery from multiorgan failure. The average follow-up duration was 17±14.5 months (range 2-42) in the 10 survivors. The remaining patients recovered and none experienced stent-graft thrombosis, stroke, or peripheral artery embolism during follow-up. CONCLUSION: A procedure that combines sandwich/periscope grafting with TEVAR, wrapping of the aorta, and supra-arch bypass grafting can be used to treat patients with acute TAAD.

Forkhead box O3 (FoxO3) regulates kidney tubular autophagy following urinary tract obstruction.

Autophagy has been shown to be important for normal homeostasis and adaptation to stress in the kidney. Yet, the molecular mechanisms regulating renal epithelial autophagy are not fully understood. Here, we explored the role of the stress-responsive transcription factor forkhead box O3 (FoxO3) in mediating injury-induced proximal tubular autophagy in mice with unilateral ureteral obstruction (UUO). We show that following UUO, FoxO3 is activated and displays nuclear expression in the hypoxic proximal tubules exhibiting high levels of autophagy. Activation of FoxO3 by mutating phosphorylation sites to enhance its nuclear expression induces profound autophagy in cultured renal epithelial cells. Conversely, deleting FoxO3 in mice results in fewer numbers of autophagic cells in the proximal tubules and reduced ratio of the autophagy-related protein LC3-II/I in the kidney post-UUO. Interestingly, autophagic cells deficient in FoxO3 contain lower numbers of autophagic vesicles per cell. Analyses of individual cells treated with autophagic inhibitors to sequentially block the autophagic flux suggest that FoxO3 stimulates the formation of autophagosomes to increase autophagic capacity but has no significant effect on autophagosome-lysosome fusion or autolysosomal clearance. Furthermore, in kidneys with persistent UUO for 7 days, FoxO3 activation increases the abundance of mRNA and protein levels of the core autophagy-related (Atg) proteins including Ulk1, Beclin-1, Atg9A, Atg4B, and Bnip3, suggesting that FoxO3 may function to maintain components of the autophagic machinery that would otherwise be consumed during prolonged autophagy. Taken together, our findings indicate that FoxO3 activation can both induce and maintain autophagic activities in renal epithelial cells in response to injury from urinary tract obstruction.

A Modified and Less Invasive Procedure in Aortic Arch Replacement.

Circulation arrest is widely used during total aortic arch replacement. However, the conventional procedures have high morbidity and mortality. We modified the hybrid procedure by optimizing the sequence of anastomosis between the graft and aorta. As a result, the time of extracorporeal circulation was obviously shortened, and cardiac arrest was needed only during the two anastomoses of the aorta with aorta graft main body. In addition, both circulation arrest and deep hypothermia were avoided. Leakage was also prevented by creating a lengthened sealing zone for stent graft deployment.

Hedgehog signaling indirectly affects tubular cell survival after obstructive kidney injury.

Hedgehog (Hh) is an evolutionary conserved signaling pathway that has important functions in kidney morphogenesis and adult organ maintenance. Recent work has shown that Hh signaling is reactivated in the kidney after injury and is an important mediator of progressive fibrosis. Pericytes and fibroblasts have been proposed to be the principal cells that respond to Hh ligands, and pharmacological attenuation of Hh signaling has been considered as a possible treatment for fibrosis, but the effect of Hh inhibition on tubular epithelial cells after kidney injury has not been reported. Using genetically modified mice in which tubule-derived hedgehog signaling is increased and mice in which this pathway is conditionally suppressed in pericytes that express the proteoglycan neuron glial protein 2 (NG2), we found that suppression of Hh signaling is associated with decreased macrophage infiltration and tubular proliferation but also increased tubular apoptosis, an effect that correlated with the reduction of tubular ß-catenin activity. Collectively, our data suggest a complex function of hedgehog signaling after kidney injury in initiating both reparative and proproliferative, prosurvival processes.