Arthur M. Lee

Background: Characterizing gene–metabolite–cardiac associations in childhood chronic kidney disease (CKD) has the potential to identify new research directions or therapeutic targets for mitigating the excessive cardiovascular risk in childhood CKD. Methods: The Chronic Kidney Disease in Children (CKiD) is a prospective cohort that recruited children with eGFR 30–90 ml/min/1.73 m ² across 54 North American centers. Predictor and outcome variables included genotypes, metabolite levels, left ventricular mass index (LVMI) z-score, and left ventricular filling pressure (E/e’). Multivariable linear regression analyses assessed metabolite associations with either LVMI z-score or E/e’, adjusting for age, sex, CKD duration, glomerular etiology, renin-angiotensin-aldosterone-system blockade, hypertension, anemia, dyslipidemia, eGFR, and proteinuria. Significance was based on the Benjamini–Hochberg False Discovery Rate p < 1.25E-8 ( p < [5E-8]/4, Bonferroni-corrected genome-wide significance threshold). Conclusions: We identified metabolites associated with cardiac markers in children with CKD. We identified genetic associations with variability in these metabolites. Collectively, these gene–metabolite associations suggest roles for inflammation, increased oxidative stress, mitochondrial and energy dysmetabolism, and gut microbial dysbiosis in mediating cardiac pathology in childhood CKD.

IMPORTANCE: Patients with acute respiratory distress syndrome (ARDS) are at risk for higher morbidity and mortality with concurrent acute kidney injury (AKI). AKI diagnosis relies on serum creatinine, which is confounded by fluid overload. Use of fluid overload-adjusted creatinine can diagnose AKI (termed Cryptic AKI) that would otherwise be missed by the traditional Kidney Disease Improving Global Outcomes (KDIGO) definition. It is unknown whether Cryptic AKI is biochemically distinct from patients without AKI. OBJECTIVES: Determine whether pediatric ARDS patients with Cryptic AKI have a biochemical profile that is similar or distinct from patients with KDIGO-defined AKI or no AKI. DESIGN, SETTING, AND PARTICIPANTS: Observational study of 333 pediatric ARDS patients in a quaternary PICU. MAIN OUTCOMES AND MEASURES: Mortality, length of stay, and ventilator-free days were measured for all patients. Biomarkers indicative of tissue damage and inflammation were measured on days 1, 3, and 7 of ARDS. RESULTS: Cryptic AKI was associated with higher mortality and longer length of stay than no AKI, similar to KDIGO AKI. Biomarkers angiopoietin-2, pro-collagen III N-terminal peptide, heat shock protein 70, tumor necrosis factor receptor 1, and interleukin-8 were significantly higher in Cryptic and KDIGO AKI than in no AKI. CONCLUSIONS AND RELEVANCE: Cryptic AKI is clinically significant and represents true kidney injury, with a biomarker profile similar to KDIGO AKI and distinct from patients without AKI.

Key Points: Given the role of diet in kids and micronutrients as cofactors, dietary micronutrient intake and its effect on the metabolome merit further study. Many children with CKD have intake below dietary reference intake of at least one micronutrient despite normal body mass index. Dietary intake of vitamins D and B12 had the greatest associations with plasma metabolites, particularly belonging to lipid pathways. Background: Children with CKD experience poor growth and development through multiple mechanisms. We aimed to describe deficiencies in dietary micronutrient intake and associate dietary micronutrient intake with metabolic pathways. Methods: The CKD in Children cohort study enrolled participants 6 months to 16 years with CKD stages 2–4 across North America. Micronutrient intake (three trace minerals, eight water-soluble vitamins, and four fat-soluble vitamins) was quantified by food frequency questionnaires at 6-month, 2-year and 4-year visits. Blood was collected at those time points for untargeted metabolomic profiling. Associations between 990 plasma metabolite features and dietary micronutrient intake were quantified with multiple linear regression using mixed effects regression models. Results: There were 575 participants with available data at the 6-month visit (mean age 12 years, 39% female, eGFR 53 ml/min per 1.73 m ² , body mass index Z-score 0.7). The most common deficiency of micronutrient intake was vitamin D (90% of participants), followed by vitamin E (77%), and more than 45% of participants did not meet the dietary reference intake of iron and folate. Children with lower eGFR had lower intake of zinc and vitamins B5 and B6. Micronutrient intake was associated with 99 total and 42 unique metabolites. Dietary intake of vitamins D and B12 was associated with many metabolites involved in lipid superpathways and phosphatidylcholine, plasmalogen, and lysophospholipid subpathways. Dietary intake of iron, folate, and vitamins C, A, and K was associated with metabolites primarily belonging to the xenobiotic, cofactor/vitamin, and amino acid superpathways. The strongest associations between dietary micronutrient intake and plasma metabolites were between intake of vitamin C with stachydrine and 3-hydroxystachydrine. Conclusions: Most children with CKD have intake below dietary reference intake of at least one micronutrient despite normal body mass index, which was associated with alterations in lipid metabolism. Clinical Trial registry name and registration number: The validation cohort, “Vitamin D Supplementation in Glomerular Disease (D-FINE),” was registered with clinicaltrials.gov, NCT01835639.

Background The gut–kidney axis is implicated in chronic kidney disease (CKD) morbidity. We describe how a panel of gut microbiome–derived toxins relates to kidney function and neurocognitive outcomes in children with CKD, consisting of indoleacetate, 3-indoxylsulfate, p-cresol glucuronide, p-cresol sulfate, and phenylacetylglutamine. Methods The Chronic Kidney Disease in Children (CKiD) cohort is a North American multicenter prospective cohort that enrolled children aged 6 months to 16 years with estimated glomerular filtration rate (eGFR) 30–89 ml/min/1.73 m². Data from the 2-year study visit were used for this analysis. Toxin quantification (Metabolon Inc., Durham, NC) was performed with ultra-high performance liquid chromatography/tandem mass spectrometry. Executive function and echocardiograms were assessed. Regression analysis examined the association of toxin levels with eGFR, CKD etiology, and neurocognitive and cardiac assessments (adjusted for age, sex, and urine protein:creatinine [UPCR]). Results There were 150 CKiD participants included in this study. All toxins levels were significantly inversely correlated with eGFR (Spearman’s rho − 0.45 to − 0.69). Children with non-glomerular CKD had significantly higher levels of 3-indoxylsulfate, phenylacetylglutamine, and p-cresol glucuronide. The toxin levels did not associate with neurocognitive outcomes. P-cresol glucuronide and phenylacetylglutamine negatively associated with left ventricular mass index z score, but did not associate with left ventricular hypertrophy. Conclusions Children with CKD have high levels of circulating gut microbiome–derived toxins. The levels of these toxins are strongly correlated with eGFR. There appear to be differences in toxin level based on glomerular versus non-glomerular etiology, even when accounting for the differences in eGFR between these two subgroups. In this sample, we did not detect any associations between these toxin levels and neurocognitive or cardiac outcomes. Graphical Abstract A higher resolution version of the Graphical abstract is available as Supplementary information

Key Points: Longitudinal untargeted metabolomics. Children with CKD have a circulating metabolome that changes over time. Background: Understanding plasma metabolome patterns in relation to changing kidney function in pediatric CKD is important for continued research for identifying novel biomarkers, characterizing biochemical pathophysiology, and developing targeted interventions. There are a limited number of studies of longitudinal metabolomics and virtually none in pediatric CKD. Methods: The CKD in Children study is a multi-institutional, prospective cohort that enrolled children aged 6 months to 16 years with eGFR 30–90 ml/min per 1.73 m². Untargeted metabolomics profiling was performed on plasma samples from the baseline, 2-, and 4-year study visits. There were technologic updates in the metabolomic profiling platform used between the baseline and follow-up assays. Statistical approaches were adopted to avoid direct comparison of baseline and follow-up measurements. To identify metabolite associations with eGFR or urine protein-creatinine ratio (UPCR) among all three time points, we applied linear mixed-effects (LME) models. To identify metabolites associated with time, we applied LME models to the 2- and 4-year follow-up data. We applied linear regression analysis to examine associations between change in metabolite level over time (∆level) and change in eGFR (∆eGFR) and UPCR (∆UPCR). We reported significance on the basis of both the false discovery rate (FDR) P < 0.05. Results: There were 1156 person-visits (N: baseline=626, 2-year=254, 4-year=276) included. There were 622 metabolites with standardized measurements at all three time points. In LME modeling, 406 and 343 metabolites associated with eGFR and UPCR at FDR n=123), we report 35 metabolites with ∆level–∆eGFR associations significant at FDR P < 0.05 and associations with UPCR in LME modeling at FDR <0.05. Conclusions: We characterized longitudinal plasma metabolomic patterns associated with eGFR and UPCR in a large pediatric CKD population. Many of these metabolite signals have been associated with CKD progression, etiology, and proteinuria in previous CKD Biomarkers Consortium studies. There were also novel metabolite associations with eGFR and proteinuria detected.

Background: Children with CKD are at risk for impaired neurocognitive functioning. We investigated metabolomic associations with neurocognition in children with CKD. Methods: We leveraged data from the Chronic Kidney Disease in Children (CKiD) study and the Neurocognitive Assessment and Magnetic Resonance Imaging Analysis of Children and Young Adults with Chronic Kidney Disease (NiCK) study. CKiD is a multi-institutional cohort that enrolled children aged 6 months to 16 years with eGFR 30–90 ml/min per 1.73 m² (n=569). NiCK is a single-center cross-sectional study of participants aged 8–25 years with eGFR2 (n=60) and matched healthy controls (n=67). Untargeted metabolomic quantification was performed on plasma (CKiD, 622 metabolites) and serum (NiCK, 825 metabolites) samples. Four neurocognitive domains were assessed: intelligence, attention regulation, working memory, and parent ratings of executive function. Repeat assessments were performed in CKiD at 2-year intervals. Linear regression and linear mixed-effects regression analyses adjusting for age, sex, delivery history, hypertension, proteinuria, CKD duration, and glomerular versus nonglomerular diagnosis were used to identify metabolites associated with neurocognitive z-scores. Analyses were performed with and without adjustment for eGFR. Results: There were multiple metabolite associations with neurocognition observed in at least two of the analytic samples (CKiD baseline, CKiD follow-up, and NiCK CKD). Most of these metabolites were significantly elevated in children with CKD compared with healthy controls in NiCK. Notable signals included associations with parental ratings of executive function: phenylacetylglutamine, indoleacetylglutamine, and trimethylamine N-oxide—and with intelligence: γ-glutamyl amino acids and aconitate. Conclusions: Several metabolites were associated with neurocognitive dysfunction in pediatric CKD, implicating gut microbiome–derived substances, mitochondrial dysfunction, and altered energy metabolism, circulating toxins, and redox homeostasis. Podcast: This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/CJASN/2023_11_17_CJN0000000000000318.mp3

Background: Untargeted plasma metabolomic profiling combined with machine learning (ML) may lead to discovery of metabolic profiles that inform our understanding of pediatric CKD causes. We sought to identify metabolomic signatures in pediatric CKD based on diagnosis: FSGS, obstructive uropathy (OU), aplasia/dysplasia/hypoplasia (A/D/H), and reflux nephropathy (RN). Methods: Untargeted metabolomic quantification (GC-MS/LC-MS, Metabolon) was performed on plasma from 702 Chronic Kidney Disease in Children study participants (n: FSGS=63, OU=122, A/D/H=109, and RN=86). Lasso regression was used for feature selection, adjusting for clinical covariates. Four methods were then applied to stratify significance: logistic regression, support vector machine, random forest, and extreme gradient boosting. ML training was performed on 80% total cohort subsets and validated on 20% holdout subsets. Important features were selected based on being significant in at least two of the four modeling approaches. We additionally performed pathway enrichment analysis to identify metabolic subpathways associated with CKD cause. Results: ML models were evaluated on holdout subsets with receiver-operator and precision-recall area-under-the-curve, F1 score, and Matthews correlation coefficient. ML models outperformed no-skill prediction. Metabolomic profiles were identified based on cause. FSGS was associated with the sphingomyelin-ceramide axis. FSGS was also associated with individual plasmalogen metabolites and the subpathway. OU was associated with gut microbiome–derived histidine metabolites. Conclusion: ML models identified metabolomic signatures based on CKD cause. Using ML techniques in conjunction with traditional biostatistics, we demonstrated that sphingomyelin-ceramide and plasmalogen dysmetabolism are associated with FSGS and that gut microbiome–derived histidine metabolites are associated with OU.

Background CEA (CEACAM5) is a well-validated cell-surface antigen that is highly expressed in multiple solid tumors. Bolt's immune-stimulating antibody conjugates (ISACs) direct a TLR7/8 agonist into tumors to activate tumor-infiltrating myeloid cells and initiate a broad innate and adaptive anti-tumor immune response.¹ The favorable properties of CEA, including robust cell surface expression, low internalization rate, and limited normal tissue expression, suggest that the antigen may be a suitable ISAC target. We are evaluating an anti-CEA ISAC, BDC-2034, as a multi-functional approach to treat CEA-expressing cancers. Methods Anti-CEA antibodies were tested for binding affinity and specificity, CEA-targeted antibody-dependent cellular phagocytosis (ADCP), and myeloid-mediated tumor cell killing. Selected antibodies were conjugated to proprietary TLR7/8 agonists, and the resulting CEA ISACs were evaluated for in vitro myeloid activation and in vivo efficacy against xenograft tumors. Results Antibody CEA1 binds to the CEA protein with high affinity (EC50 = 0.25 nM), binds selectively to CEA-positive tumor cell lines, and mediates ADCP more efficiently than a reference anti-CEA antibody, labetuzumab (figure 1). We generated BDC-2034 by conjugating a potent TLR7/8 agonist to CEA1. BDC-2034 tumor cell binding drives myeloid effector cell ADCP, agonist delivery to TLR7 and TLR8 in endosomes, and secretion of cytokines critical for innate and adaptive immunity (including IL-12p70, CXCL10, and TNFa). In the HPAF II + cDC co-culture model, IL-12p70 is induced with EC50 = 1.2 nM, and the level of induction is at least ten-fold higher than with ISACs using labetuzumab (figure 2). Potent cellular activity is strictly dependent on tumor cell CEA expression; in whole blood, in the absence of CEA-expressing tumor cells, cytokine induction was only observed at approximately 100-fold higher concentrations. BDC-2034 inhibits the growth of HPAF II xenograft tumors in SCID/beige mice with a minimal efficacious dose (MED) of 1 mg/kg, demonstrating anti-tumor activity solely through innate immune activation (figure 3). The TLR7/8 agonist in BDC-2034 has relatively poor activity in mice; a surrogate CEA1 ISAC with a mouse TLR7-activating agonist achieved MED = 0.5 mg/kg in the HPAF II model, with eradication of all tumors at the 5 mg/kg dose. Conclusions These pre-clinical data demonstrate the potential of BDC-2034 to treat CEA-expressing human cancers. Most importantly, the antigen-dependent induction of immune-stimulating cytokines promises a robust immune response that combines the activation of innate and adaptive arms. Reference Ackerman S, Pearson C, Gregorio J. Immune-stimulating antibody conjugates elicit robust myeloid activation and durable antitumor immunity. Nature Cancer 2021;2:18–33. https://doi.org/10.1038/s43018-020-00136-x ADCP. Anti-CEA antibody CEA1 is an efficient inducer of ADCP of Raji/CEA cells by M-CSF differentiated monocyte-derived macrophages Tumor-dependent dendritic cell activation. BDC-2034 induces IL-12p70 secretion from primary dendritic cells (cDC); native CEA1 antibody and reference anti-CEA ISAC are ineffective Efficacy against xenograft tumors. BDC-2034 inhibits the growth of HPAF II tumors in SCID/beige mice; native CEA1 antibody and isotype ISAC are ineffective

Background PD-L1 is an immune checkpoint that regulates anti-tumor T cell responses and is expressed on tumor cells as well as tumor-infiltrating immune cells across many tumor types. Immune-stimulating antibody conjugates (ISACs) consist of tumor-targeting antibodies conjugated to immune stimulants and are designed to activate the innate and adaptive immune systems against tumor cells following systemic administration. Here we show that PD-L1-targeted TLR7/8 ISACs elicit robust myeloid cell activation which leads to improved anti-tumor responses compared to anti-PD-L1 treatment in pre-clinical tumor models. Methods A panel of proprietary anti-PD-L1 antibodies was identified through a phage display screen and subsequently tested for PD-L1 binding affinity and specificity, PD-L1/PD-1 blocking, antibody-dependent cellular phagocytosis (ADCP) by myeloid cells, and anti-tumor efficacy. Lead antibodies were conjugated to proprietary TLR7/8 agonists, and the resulting PD-L1 ISACs were evaluated for in vitro myeloid cell activation and in vivo efficacy against syngeneic and xenograft tumors. Results Anti-PD-L1 antibodies induced robust ADCP by myeloid effector cells and medium to strong PD-L1/PD-1 blockade in vitro. Selected antibodies inhibited the growth of syngeneic MC38-hPD-L1 tumors in vivo, confirming efficient immune-checkpoint blockade. The conjugated PD-L1 ISACs induced robust, target-dependent activation of myeloid cells when co-cultured with PD-L1-expressing tumor cells, as measured by increased secretion of such cytokines as IL-12p70, IFN-alpha, and TNF-alpha. Importantly, myeloid activation was observed following co-culture with tumor cells having various levels of endogenous PD-L1 expression that was within the range of PD-L1 expression observed in human tumors. Systemically administered surrogate PD-L1 ISACs were well tolerated in mice and showed improved anti-tumor efficacy over anti-PD-L1 antibodies, with significant tumor growth delay or complete responses frequently observed in syngeneic (e.g. MB49, MC38-hPD-L1) as well as xenograft (e.g. HCC1954-hPD-L1) tumor models. Conclusions These data demonstrate the potential of a PD-L1-targeted ISAC as a multifunctional therapeutic that may improve efficacy of PD-L1/PD-1 inhibition by combining three mechanisms of action into a single molecule: TLR-mediated myeloid cell activation, T cell activation through immune-checkpoint inhibition as well as ADCP. Ethics Approval All animal studies were performed in accordance with Institutional Animal Care and Use Committee (IACUC)-approved protocols.