Thrombotic Microangiopathies and the Kidney.

Thrombotic microangiopathy (TMA) is a pathological lesion that occurs due to endothelial injury. It can be seen in a heterogenous group of disorders, typically characterized by microangiopathic hemolytic anemia, thrombocytopenia, and end-organ ischemia. TMA can also be renal limited with no systemic manifestations. There are multiple etiologies of a TMA with complement activation being a core underlying mechanism, although the nature and extent of complement involvement can vary. A further complicated factor is the cross talk between complement, neutrophils, and coagulation pathways in the pathophysiology of TMAs. Therefore, a thorough and systematic clinical history and laboratory evaluation are critical to establish the cause and pathophysiology of a TMA. Furthermore, TMAs are associated with significant morbidity and mortality, and timely diagnosis is key for appropriate management and to prevent end-stage kidney disease and other associated complications. In this review, we focus on the pathology, mechanisms, diagnostic work up and treatment of TMAs associated with various etiologies. We also define the complement evaluations that should be conducted in these patients and further highlight the currently approved complement therapies as well as others in the pipeline.

Microbial influences on severity and sex bias of systemic autoimmunity.

Commensal microbes have the capacity to affect development and severity of autoimmune diseases. Germ-free (GF) animals have proven to be a fine tool to obtain definitive answers to the queries about the microbial role in these diseases. Moreover, GF and gnotobiotic animals can be used to dissect the complex symptoms and determine which are regulated (enhanced or attenuated) by microbes. These include disease manifestations that are sex biased. Here, we review comparative analyses conducted between GF and Specific-Pathogen Free (SPF) mouse models of autoimmunity. We present data from the B6;NZM-Sle1NZM2410/AegSle2NZM2410/AegSle3NZM2410/Aeg-/LmoJ (B6.NZM) mouse model of systemic lupus erythematosus (SLE) characterized by multiple measurable features. We compared the severity and sex bias of SPF, GF, and ex-GF mice and found variability in the severity and sex bias of some manifestations. Colonization of GF mice with the microbiotas taken from B6.NZM mice housed in two independent institutions variably affected severity and sexual dimorphism of different parameters. Thus, microbes regulate both the severity and sexual dimorphism of select SLE traits. The sensitivity of particular trait to microbial influence can be used to further dissect the mechanisms driving the disease. Our results demonstrate the complexity of the problem and open avenues for further investigations.

Promises, Pitfalls, and Clinical Applications of Artificial Intelligence in Pediatrics.

Artificial intelligence (AI) broadly describes a branch of computer science focused on developing machines capable of performing tasks typically associated with human intelligence. Those who connect AI with the world of science fiction may meet its growing rise with hesitancy or outright skepticism. However, AI is becoming increasingly pervasive in our society, from algorithms helping to sift through airline fares to substituting words in emails and SMS text messages based on user choices. Data collection is ongoing and is being leveraged by software platforms to analyze patterns and make predictions across multiple industries. Health care is gradually becoming part of this technological transformation, as advancements in computational power and storage converge with the rapid expansion of digitized medical information. Given the growing and inevitable integration of AI into health care systems, it is our viewpoint that pediatricians urgently require training and orientation to the uses, promises, and pitfalls of AI in medicine. AI is unlikely to solve the full array of complex challenges confronting pediatricians today; however, if used responsibly, it holds great potential to improve many aspects of care for providers, children, and families. Our aim in this viewpoint is to provide clinicians with a targeted introduction to the field of AI in pediatrics, including key promises, pitfalls, and clinical applications, so they can play a more active role in shaping the future impact of AI in medicine.

Pseudo-spectral angle mapping for automated pixel-level analysis of highly multiplexed tissue image data.

The rapid development of highly multiplexed microscopy systems has enabled the study of cells embedded within their native tissue, which is providing exciting insights into the spatial features of human disease [1]. However, computational methods for analyzing these high-content images are still emerging, and there is a need for more robust and generalizable tools for evaluating the cellular constituents and underlying stroma captured by high-plex imaging [2]. To address this need, we have adapted spectral angle mapping - an algorithm used widely in hyperspectral image analysis - to compress the channel dimension of high-plex immunofluorescence images. As many high-plex immunofluorescence imaging experiments probe unique sets of protein markers, existing cell and pixel classification models do not typically generalize well. Pseudospectral angle mapping (pSAM) uses reference pseudospectra - or pixel vectors - to assign each pixel in an image a similarity score to several cell class reference vectors, which are defined by each unique staining panel. Here, we demonstrate that the class maps provided by pSAM can directly provide insight into the prevalence of each class defined by reference pseudospectra. In a dataset of high-plex images of colon biopsies from patients with gut autoimmune conditions, sixteen pSAM class representation maps were combined with instance segmentation of cells to provide cell class predictions. Finally, pSAM detected a diverse set of structure and immune cells when applied to a novel dataset of kidney biopsies imaged with a 43-marker panel. In summary, pSAM provides a powerful and readily generalizable method for evaluating high-plex immunofluorescence image data.

An International Survey of Genitourinary and Renal Pathologists Regarding Evaluation of the Non-Neoplastic Parenchyma in Kidney Cancer Specimens.

Increasing survivorship in kidney cancer patients has shifted treatment strategies to optimize renal function preservation. In 2010, the College of American Pathologists (CAP) updated their synoptic reporting guidelines for tumor nephrectomies to require evaluation of the nonneoplastic kidney parenchyma. We conducted this study to understand current practice behaviors regarding the evaluation of the nonneoplastic kidney parenchyma in tumor nephrectomy specimens. We emailed a 14-item multiple-choice survey to members of the Renal Pathology Society and Genitourinary Pathology Society. We also emailed a 12-item survey to program and associate program directors of American pathology residencies to assess the current state of renal pathology education. Ninety-eight genitourinary and 104 renal pathologists responded to the survey on the nonneoplastic kidney parenchyma. Ninety-five percent of respondents who examine tumor nephrectomies reported evaluating the nonneoplastic kidney parenchyma. Seventy-five percent of genitourinary pathologists and 67% of renal pathologists use synoptic reporting, and 81% use the CAP protocol. Thirty-nine percent of respondents report always contacting the clinician when they find evidence of medical renal disease. Forty-two program leaders responded to our renal pathology education survey, and 64% of them have a mandatory renal pathology rotation that on average lasts about 2 to 4 weeks. The majority of pathologists examine the nonneoplastic kidney parenchyma of tumor nephrectomies and frequently report incidences of new medical renal disease directly to clinicians, but there remains room for improvement and educational gaps during residency training. Further efforts to standardize both this evaluation and renal pathology education will improve patient care.

The Detrimental Impact of End-Stage Kidney Disease Is Not Reflected in Autopsy Reports.

CONTEXT.­: End-stage kidney disease (ESKD) is defined as renal impairment requiring renal replacement therapy to sustain life. With a 1-year mortality of ∼20% to 30%, many die of complications related to this disease. OBJECTIVE.­: To determine the percentage of autopsy cases of decedents with ESKD in which the contribution of ESKD to death is accurately reflected in the final report. DESIGN.­: Autopsy case records were retrospectively reviewed at 4 institutions (Yale New Haven Hospital, University of Chicago Medical Center, University of Illinois at Chicago Hospital, University of Iowa Hospital). Clinical, macroscopic, and microscopic autopsy findings were reviewed, with attention to renal disease findings. RESULTS.­: One hundred sixty decedents with documented ESKD and premortem dialysis who underwent autopsy assessment were identified. ESKD was implicated as a cause of death (CoD) or significant contributing factor in 44 cases (28%), but not in the remaining 116 cases (72%). Cardiovascular disease was the most common CoD in ESKD. There was significant interpathologist variation in the inclusion of ESKD as a CoD across institutions. These rates ranged from 85% correlation (23 of 27 cases), to 13% (4 of 31 and 8 of 62 cases at 2 institutions), and 22.5% (9 of 40 cases) across the 4 participating institutions. CONCLUSIONS.­: The recognition at autopsy of ESKD as a CoD or contributing CoD at autopsy in patients undergoing dialysis remains low (28%). The detrimental impact of ESKD is not reflected in hospital autopsy reports, which carries implications for collection of vital statistics and allocation of research funding for kidney diseases.

Podocyte injury at young age causes premature senescence and worsens glomerular aging.

As life expectancy continues to rise, age-related diseases are becoming more prevalent. For example, proteinuric glomerular diseases typified by podocyte injury have worse outcomes in the elderly compared with young patients. However, the reasons are not well understood. We hypothesized that injury to nonaged podocytes induces senescence, which in turn augments their aging processes. In primary cultured human podocytes, injury induced by a cytopathic antipodocyte antibody, adriamycin, or puromycin aminonucleoside increased the senescence-related genes CDKN2A (p16INK4a/p14ARF), CDKN2D (p19INK4d), and CDKN1A (p21). Podocyte injury in human kidney organoids was accompanied by increased expression of CDKN2A, CDKN2D, and CDKN1A. In young mice, experimental focal segmental glomerulosclerosis (FSGS) induced by adriamycin and antipodocyte antibody increased the glomerular expression of p16, p21, and senescence-associated ß-galactosidase (SA-ß-gal). To assess the long-term effects of early podocyte injury-induced senescence, we temporally followed young mice with experimental FSGS through adulthood (12 m of age) and middle age (18 m of age). p16 and Sudan black staining were higher at middle age in mice with earlier FSGS compared with age-matched mice that did not get FSGS when young. This was accompanied by lower podocyte density, reduced canonical podocyte protein expression, and increased glomerular scarring. These results are consistent with injury-induced senescence in young podocytes, leading to increased senescence of podocytes by middle age accompanied by lower podocyte lifespan and health span.NEW & NOTEWORTHY Glomerular function is decreased by aging. However, little is known about the molecular mechanisms involved in age-related glomerular changes and which factors could contribute to a worse glomerular aging process. Here, we reported that podocyte injury in young mice and culture podocytes induced senescence, a marker of aging, and accelerates glomerular aging when compared with healthy aging mice.

Prospective observational study to validate a next-generation sequencing blood RNA signature to predict early kidney transplant rejection.

The objective of this study was to validate the performance of Tutivia, a peripheral blood gene expression signature, in predicting early acute rejection (AR) post-kidney transplant. Recipients of living or deceased donor kidney transplants were enrolled in a nonrandomized, prospective, global, and observational study (NCT04727788). The main outcome was validation of the area under the curve (AUC) of Tutivia vs serum creatinine at biopsy alone, or Tutivia + serum creatinine at biopsy. Of the 151 kidney transplant recipients, the mean cohort age was 53 years old, and 64% were male. There were 71% (107/151) surveillance/protocol biopsies and 29% (44/151) for-cause biopsies, with a 31% (47/151) overall rejection rate. Tutivia (AUC 0.69 [95% CI: 0.59-0.77]) and AUC of Tutivia + creatinine at biopsy (0.68 [95% CI: 0.59-0.77]) were greater than the AUC of creatinine at biopsy alone (0.51.4 [95% CI: 0.43-0.60]). Applying a model cut-off of 50 (scale 0-100) generated a high- and low-risk category for AR with a negative predictive value of 0.79 (95% CI: 0.71-0.86), a positive predictive value of 0.60 (95% CI: 0.45-0.74), and an odds ratio of 5.74 (95% CI: 2.63-12.54). Tutivia represents a validated noninvasive approach for clinicians to accurately predict early AR, beyond the current standard of care.

Implantable bioelectronic systems for early detection of kidney transplant rejection.

Early-stage organ transplant rejection can be difficult to detect. Percutaneous biopsies occur infrequently and are risky, and measuring biomarker levels in blood can lead to false-negative and -positive outcomes. We developed an implantable bioelectronic system capable of continuous, real-time, long-term monitoring of the local temperature and thermal conductivity of a kidney for detecting inflammatory processes associated with graft rejection, as demonstrated in rat models. The system detects ultradian rhythms, disruption of the circadian cycle, and/or a rise in kidney temperature. These provide warning signs of acute kidney transplant rejection that precede changes in blood serum creatinine/urea nitrogen by 2 to 3 weeks and approximately 3 days for cases of discontinued and absent administration of immunosuppressive therapy, respectively.

Inhibiting NLRP3 signaling in aging podocytes improves their life- and health-span.

The decrease in the podocyte's lifespan and health-span that typify healthy kidney aging cause a decrease in their normal structure, physiology and function. The ability to halt and even reverse these changes becomes clinically relevant when disease is superimposed on an aged kidney. RNA-sequencing of podocytes from middle-aged mice showed an inflammatory phenotype with increases in the NLRP3 inflammasome, signaling for IL2/Stat5, IL6 and TNF, interferon gamma response, allograft rejection and complement, consistent with inflammaging. Furthermore, injury-induced NLRP3 signaling in podocytes was further augmented in aged mice compared to young ones. The NLRP3 inflammasome (NLRP3, Caspase-1, IL1ß IL-18) was also increased in podocytes of middle-aged humans. Higher transcript expression for NLRP3 in human glomeruli was accompanied by reduced podocyte density and increased global glomerulosclerosis and glomerular volume. Pharmacological inhibition of NLRP3 with MCC950, or gene deletion, reduced podocyte senescence and the genes typifying aging in middle-aged mice, which was accompanied by an improved podocyte lifespan and health-span. Moreover, modeling the injury-dependent increase in NLRP3 signaling in human kidney organoids confirmed the anti-senescence effect of MC9950. Finally, NLRP3 also impacted liver aging. Together, these results suggest a critical role for the NLRP3 inflammasome in podocyte and liver aging.

Molecular architecture of proliferative lupus nephritis as elucidated using 50-plex imaging mass cytometry proteomics.

Due to unique advantages that allow high-dimensional tissue profiling, we postulated imaging mass cytometry (IMC) may shed novel insights on the molecular makeup of proliferative lupus nephritis (LN). This study interrogates the spatial expression profiles of 50 target proteins in LN and control kidneys. Proliferative LN glomeruli are marked by podocyte loss with immune infiltration dominated by CD45RO+, HLA-DR+ memory CD4 and CD8 T-cells, and CD163+ macrophages, with similar changes in tubulointerstitial regions. Macrophages are the predominant HLA-DR expressing antigen presenting cells with little expression elsewhere, while macrophages and T-cells predominate cellular crescents. End-stage sclerotic glomeruli are encircled by an acellular fibro-epithelial Bowman's space surrounded by immune infiltrates, all enmeshed in fibronectin. Proliferative LN also shows signs indicative of epithelial to mesenchymal plasticity of tubular cells and parietal epithelial cells. IMC enabled proteomics is a powerful tool to delineate the spatial architecture of LN at the protein level.

Group 2 innate lymphoid cells constrain type 3/17 lymphocytes in shared stromal niches to restrict liver fibrosis.

Group 2 innate lymphoid cells (ILC2s) cooperate with adaptive Th2 cells as key organizers of tissue type 2 immune responses, while a spectrum of innate and adaptive lymphocytes coordinate early type 3/17 immunity. Both type 2 and type 3/17 lymphocyte associated cytokines are linked to tissue fibrosis, but how their dynamic and spatial topographies may direct beneficial or pathologic organ remodelling is unclear. Here we used volumetric imaging in models of liver fibrosis, finding accumulation of periportal and fibrotic tract IL-5 + lymphocytes, predominantly ILC2s, in close proximity to expanded type 3/17 lymphocytes and IL-33 high niche fibroblasts. Ablation of IL-5 + lymphocytes worsened carbon tetrachloride-and bile duct ligation-induced liver fibrosis with increased niche IL-17A + type 3/17 lymphocytes, predominantly γδ T cells. In contrast, concurrent ablation of IL-5 + and IL-17A + lymphocytes reduced this progressive liver fibrosis, suggesting a cross-regulation of type 2 and type 3 lymphocytes at specialized fibroblast niches that tunes hepatic fibrosis.

An Introduction to Artificial Intelligence in Developmental and Behavioral Pediatrics.

ABSTRACT: Technological breakthroughs, together with the rapid growth of medical information and improved data connectivity, are creating dramatic shifts in the health care landscape, including the field of developmental and behavioral pediatrics. While medical information took an estimated 50 years to double in 1950, by 2020, it was projected to double every 73 days. Artificial intelligence (AI)-powered health technologies, once considered theoretical or research-exclusive concepts, are increasingly being granted regulatory approval and integrated into clinical care. In the United States, the Food and Drug Administration has cleared or approved over 160 health-related AI-based devices to date. These trends are only likely to accelerate as economic investment in AI health care outstrips investment in other sectors. The exponential increase in peer-reviewed AI-focused health care publications year over year highlights the speed of growth in this sector. As health care moves toward an era of intelligent technology powered by rich medical information, pediatricians will increasingly be asked to engage with tools and systems underpinned by AI. However, medical students and practicing clinicians receive insufficient training and lack preparedness for transitioning into a more AI-informed future. This article provides a brief primer on AI in health care. Underlying AI principles and key performance metrics are described, and the clinical potential of AI-driven technology together with potential pitfalls is explored within the developmental and behavioral pediatric health context.

Upregulated PD-1 signaling antagonizes glomerular health in aged kidneys and disease.

With an aging population, kidney health becomes an important medical and socioeconomic factor. Kidney aging mechanisms are not well understood. We previously showed that podocytes isolated from aged mice exhibit increased expression of programmed cell death protein 1 (PD-1) surface receptor and its 2 ligands (PD-L1 and PD-L2). PDCD1 transcript increased with age in microdissected human glomeruli, which correlated with lower estimated glomerular filtration rate and higher segmental glomerulosclerosis and vascular arterial intima-to-lumen ratio. In vitro studies in podocytes demonstrated a critical role for PD-1 signaling in cell survival and in the induction of a senescence-associated secretory phenotype. To prove PD-1 signaling was critical to podocyte aging, aged mice were injected with anti-PD-1 antibody. Treatment significantly improved the aging phenotype in both kidney and liver. In the glomerulus, it increased the life span of podocytes, but not that of parietal epithelial, mesangial, or endothelial cells. Transcriptomic and immunohistochemistry studies demonstrated that anti-PD-1 antibody treatment improved the health span of podocytes. Administering the same anti-PD-1 antibody to young mice with experimental focal segmental glomerulosclerosis (FSGS) lowered proteinuria and improved podocyte number. These results suggest a critical contribution of increased PD-1 signaling toward both kidney and liver aging and in FSGS.

Specific in situ inflammatory states associate with progression to renal failure in lupus nephritis.

BACKGROUNDIn human lupus nephritis (LN), tubulointerstitial inflammation (TII) on biopsy predicts progression to end-stage renal disease (ESRD). However, only about half of patients with moderate-to-severe TII develop ESRD. We hypothesized that this heterogeneity in outcome reflects different underlying inflammatory states. Therefore, we interrogated renal biopsies from LN longitudinal and cross-sectional cohorts.METHODSData were acquired using conventional and highly multiplexed confocal microscopy. To accurately segment cells across whole biopsies, and to understand their spatial relationships, we developed computational pipelines by training and implementing several deep-learning models and other computer vision techniques.RESULTSHigh B cell densities were associated with protection from ESRD. In contrast, high densities of CD8+, γδ, and other CD4-CD8- T cells were associated with both acute renal failure and progression to ESRD. B cells were often organized into large periglomerular neighborhoods with Tfh cells, while CD4- T cells formed small neighborhoods in the tubulointerstitium, with frequency that predicted progression to ESRD.CONCLUSIONThese data reveal that specific in situ inflammatory states are associated with refractory and progressive renal disease.FUNDINGThis study was funded by the NIH Autoimmunity Centers of Excellence (AI082724), Department of Defense (LRI180083), Alliance for Lupus Research, and NIH awards (S10-OD025081, S10-RR021039, and P30-CA14599).

Association of Congenital and Acquired Cardiovascular Conditions With COVID-19 Severity Among Pediatric Patients in the US.

Importance: Identifying the associations between severe COVID-19 and individual cardiovascular conditions in pediatric patients may inform treatment. Objective: To assess the association between previous or preexisting cardiovascular conditions and severity of COVID-19 in pediatric patients. Design, Setting, and Participants: This retrospective cohort study used data from a large, multicenter, electronic health records database in the US. The cohort included patients aged 2 months to 17 years with a laboratory-confirmed diagnosis of COVID-19 or a diagnosis code indicating infection or exposure to SARS-CoV-2 at 85 health systems between March 1, 2020, and January 31, 2021. Exposures: Diagnoses for 26 cardiovascular conditions between January 1, 2015, and December 31, 2019 (before infection with SARS-CoV-2). Main Outcomes and Measures: The main outcome was severe COVID-19, defined as need for supplemental oxygen or in-hospital death. Mixed-effects, random intercept logistic regression modeling assessed the significance and magnitude of associations between 26 cardiovascular conditions and COVID-19 severity. Multiple comparison adjustment was performed using the Benjamini-Hochberg false discovery rate procedure. Results: The study comprised 171 416 pediatric patients; the median age was 8 years (IQR, 2-14 years), and 50.28% were male. Of these patients, 17 065 (9.96%) had severe COVID-19. The random intercept model showed that the following cardiovascular conditions were associated with severe COVID-19: cardiac arrest (odds ratio [OR], 9.92; 95% CI, 6.93-14.20), cardiogenic shock (OR, 3.07; 95% CI, 1.90-4.96), heart surgery (OR, 3.04; 95% CI, 2.26-4.08), cardiopulmonary disease (OR, 1.91; 95% CI, 1.56-2.34), heart failure (OR, 1.82; 95% CI, 1.46-2.26), hypotension (OR, 1.57; 95% CI, 1.38-1.79), nontraumatic cerebral hemorrhage (OR, 1.54; 95% CI, 1.24-1.91), pericarditis (OR, 1.50; 95% CI, 1.17-1.94), simple biventricular defects (OR, 1.45; 95% CI, 1.29-1.62), venous embolism and thrombosis (OR, 1.39; 95% CI, 1.11-1.73), other hypertensive disorders (OR, 1.34; 95% CI, 1.09-1.63), complex biventricular defects (OR, 1.33; 95% CI, 1.14-1.54), and essential primary hypertension (OR, 1.22; 95% CI, 1.08-1.38). Furthermore, 194 of 258 patients (75.19%) with a history of cardiac arrest were younger than 12 years. Conclusions and Relevance: The findings suggest that some previous or preexisting cardiovascular conditions are associated with increased severity of COVID-19 among pediatric patients in the US and that morbidity may be increased among individuals children younger than 12 years with previous cardiac arrest.

2',3',4'-Trihydroxychalcone changes estrogen receptor α regulation of genes and breast cancer cell proliferation by a reprogramming mechanism.

BACKGROUND: Menopausal hormone therapy (MHT) is recommended for only five years to treat vasomotor symptoms and vulvovaginal atrophy because of safety concerns with long-term treatment. We investigated the ability of 2',3',4'-trihydroxychalcone (2',3',4'-THC) to modulate estrogen receptor (ER)-mediated responses in order to find drug candidates that could potentially prevent the adverse effects of long-term MHT treatment. METHODS: Transfection assays, real time-polymerase chain reaction, and microarrays were used to evaluate the effects of 2',3',4'-THC on gene regulation. Radioligand binding studies were used to determine if 2',3',4'-THC binds to ERα. Cell proliferation was examined in MCF-7 breast cancer cells by using growth curves and flow cytometry. Western blots were used to determine if 2',3',4'-THC alters the E2 activation of the MAPK pathway and degradation of ERα. Chromatin immunoprecipitation was used to measure ERα binding to genes. RESULTS: The 2',3',4'-THC/E2 combination produced a synergistic activation with ERα on reporter and endogenous genes in human U2OS osteosarcoma cells. Microarrays identified 824 genes that we termed reprogrammed genes because they were not regulated in U2OS-ERα cells unless they were treated with 2',3',4'-THC and E2 at the same time. 2',3',4'-THC blocked the proliferation of MCF-7 cells by preventing the E2-induced activation of MAPK and c-MYC transcription. The antiproliferative mechanism of 2',3',4'-THC differs from selective estrogen receptor modulators (SERMs) because 2',3',4'-THC did not bind to the E2 binding site in ERα like SERMs. CONCLUSION: Our study suggests that 2',3',4'-THC may represent a new class of ERα modulators that do not act as a direct agonists or antagonists. We consider 2',3',4'-THC to be a reprogramming compound, since it alters the activity of ERα on gene regulation and cell proliferation without competing with E2 for binding to ERα. The addition of a reprogramming drug to estrogens in MHT may offer a new strategy to overcome the adverse proliferative effects of estrogen in MHT by reprogramming ERα as opposed to an antagonist mechanism that involves blocking the binding of estrogen to ERα.