Browsing by Author "Saxena, Vijay"
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Item Adolescents with urinary stones have elevated urine levels of inflammatory mediators(Springer, 2019-04-16) Kusumi, Kirsten; Ketz, John; Saxena, Vijay; Spencer, John David; Safadi, Fayez; Schwaderer, Andrew; Pediatrics, School of MedicineBackground: Urinary stone are increasing in children, primarily during adolescence. Although urinary stones are often viewed in the context of intermittent stone events, increasing evidence indicates that stones are a metabolic process associated with chronic kidney disease and low bone mass. These aforementioned stone associated conditions may have pediatric origins. Objective: To compare urine inflammatory markers in otherwise healthy stone forming children versus matched controls. Methods: Urine samples were collected from 12 adolescents with urinary stones along with 15 controls. The levels of 30 urine cytokines were measured using a Mesoscale 30-Plex Human Cytokine panel and normalized to urine creatinine levels. Results: Macrophage inflammatory protein 1β and Interleukin 13 levels were significantly elevated in the urine of the stone forming adolescents compared to controls. Interleukin 17A was elevated in the urine of controls. Conclusions: This study indicates that urine levels of cytokines involved in chronic inflammation and fibrosis are elevated urinary stone formers as early as adolescence. Because stone formers are at risk for chronic kidney disease, Macrophage inflammatory protein 1β and Interleukin 13 represent investigative targets.Item DCHS1 DNA copy number loss associated with pediatric urinary tract infection risk(Sage, 2020-08) Qureshi, Aslam H.; Liang, Dong; Canas, Jorge; Hooks, Jenaya; Arrregui, Samuel W.; Saxena, Vijay; Rooney, Robert; Nolan, Vikki; Schwaderer, Andrew L.; Hains, David S.; Pediatrics, School of MedicineUrinary tract infections (UTI), associated with vesicoureteral reflux (VUR), can lead to chronic kidney disease. Genetic alterations in the innate immune defenses contribute to UTI risk. We investigated a novel gene, Dachsous Cadherin-Related 1 ( DCHS1), in children with UTI. We determined absolute DNA copy number (CN) of DCHS1 in children with UTI. In this case-control study, we utilized multiple complementary methods to determine the genomic CN of DCHS1. Children with ( n = 370) and without ( n = 71) VUR from two well-phenotyped clinical trials of UTI were copy-typed and compared to 491 healthy controls with no known history of VUR or UTI. Less than 1% of controls had a single copy of DCHS1, while 31% of children with UTI and no VUR and 7% of children with UTI and VUR had a single copy of the DCHS1 gene. Using immunostaining, we localized expression postnatally to the bladder and renal epithelia. Mice were also challenged with two uropathogenic Escherichia coli strains, and Dchs1 mRNA was quantified. This study represents the first report of DCHS1 in association with pediatric UTI. We hypothesize that its role in innate immunity is critical to lower urinary tract defense. Further investigation is required to determine the role of DCHS1 in innate immunity.Item DCHS1 DNA copy number loss associated with pediatric urinary tract infection risk(SAGE, 2020-04-15) Qureshi, Aslam H.; Liang, Dong; Canas, Jorge; Hooks, Jenaya; Arrregui, Samuel W.; Saxena, Vijay; Rooney, Robert; Nolan, Vikki; Schwaderer, Andrew L.; Hains, David S.; Pediatrics, School of MedicineUrinary tract infections (UTI), associated with vesicoureteral reflux (VUR), can lead to chronic kidney disease. Genetic alterations in the innate immune defenses contribute to UTI risk. We investigated a novel gene, Dachsous Cadherin-Related 1 (DCHS1), in children with UTI. We determined absolute DNA copy number (CN) of DCHS1 in children with UTI. In this case-control study, we utilized multiple complementary methods to determine the genomic CN of DCHS1. Children with (n = 370) and without (n = 71) VUR from two well-phenotyped clinical trials of UTI were copy-typed and compared to 491 healthy controls with no known history of VUR or UTI. Less than 1% of controls had a single copy of DCHS1, while 31% of children with UTI and no VUR and 7% of children with UTI and VUR had a single copy of the DCHS1 gene. Using immunostaining, we localized expression postnatally to the bladder and renal epithelia. Mice were also challenged with two uropathogenic Escherichia coli strains, and Dchs1 mRNA was quantified. This study represents the first report of DCHS1 in association with pediatric UTI. We hypothesize that its role in innate immunity is critical to lower urinary tract defense. Further investigation is required to determine the role of DCHS1 in innate immunity.Item DEFA1A3 DNA gene-dosage regulates the kidney innate immune response during upper urinary tract infection(Cold Spring Harbor Laboratory, 2024-04-05) Canas, Jorge J.; Arregui, Samuel W.; Zhang, Shaobo; Knox, Taylor; Calvert, Christi; Saxena, Vijay; Schwaderer, Andrew L.; Hains, David S.; Pediatrics, School of MedicineAntimicrobial peptides (AMPs) are host defense effectors with potent neutralizing and immunomodulatory functions against invasive pathogens. The AMPs α-Defensin 1-3/DEFA1A3 participate in innate immune responses and influence patient outcomes in various diseases. DNA copy-number variations in DEFA1A3 have been associated with severity and outcomes in infectious diseases including urinary tract infections (UTIs). Specifically, children with lower DNA copy numbers were more susceptible to UTIs. The mechanism of action by which α-Defensin 1-3/DEFA1A3 copy-number variations lead to UTI susceptibility remains to be explored. In this study, we use a previously characterized transgenic knock-in of the human DEFA1A3 gene mouse to dissect α-Defensin 1-3 gene dose-dependent antimicrobial and immunomodulatory roles during uropathogenic Escherichia coli (UPEC) UTI. We elucidate the relationship between kidney neutrophil- and collecting duct intercalated cell-derived α-Defensin 1-3/DEFA1A3 expression and UTI. We further describe cooperative effects between α-Defensin 1-3 and other AMPs that potentiate the neutralizing activity against UPEC. Cumulatively, we demonstrate that DEFA1A3 directly protects against UPEC meanwhile impacting pro-inflammatory innate immune responses in a gene dosage-dependent manner.Item Deleted in malignant brain tumor 1 genetic variation confers urinary tract infection risk in children and mice(Wiley, 2021-07) Hains, David S.; Polley, Shamik; Liang, Dong; Saxena, Vijay; Arregui, Samuel; Ketz, John; Barr-Beare, Evan; Rawson, Ashley; Spencer, John D.; Cohen, Ariel; Hansen, Pernille L.; Tuttolomondo, Martina; Casella, Cinzia; Ditzel, Henrik J.; Cohen, Daniel; Hollox, Edward J.; Schwaderer, Andrew L.; Pediatrics, School of MedicineItem Human neutrophil peptides 1-3 protect the murine urinary tract from uropathogenic Escherichia coli challenge(National Academy of Science, 2022) Canas, Jorge J.; Liang, Dong; Saxena, Vijay; Hooks, Jenaya; Arregui, Samuel W.; Gao, Hongyu; Liu, Yunlong; Kish, Danielle; Linn, Sarah C.; Bdeir, Khalil; Cines, Douglas B.; Fairchild, Robert L.; Spencer, John D.; Schwaderer, Andrew L.; Hains, David S.; Pediatrics, School of MedicineAntimicrobial peptides (AMPs) are critical to the protection of the urinary tract of humans and other animals from pathogenic microbial invasion. AMPs rapidly destroy pathogens by disrupting microbial membranes and/or augmenting or inhibiting the host immune system through a variety of signaling pathways. We have previously demonstrated that alpha-defensins 1-3 (DEFA1A3) are AMPs expressed in the epithelial cells of the human kidney collecting duct in response to uropathogens. We also demonstrated that DNA copy number variations in the DEFA1A3 locus are associated with UTI and pyelonephritis risk. Because DEFA1A3 is not expressed in mice, we utilized human DEFA1A3 gene transgenic mice (DEFA4/4) to further elucidate the biological relevance of this locus in the murine urinary tract. We demonstrate that the kidney transcriptional and translational expression pattern is similar in humans and the human gene transgenic mouse upon uropathogenic Escherichia coli (UPEC) stimulus in vitro and in vivo. We also demonstrate transgenic human DEFA4/4 gene mice are protected from UTI and pyelonephritis under various UPEC challenges. This study serves as the foundation to start the exploration of manipulating the DEFA1A3 locus and alpha-defensins 1-3 expression as a potential therapeutic target for UTIs and other infectious diseases.Item Kidney intercalated cells are phagocytic and acidify internalized uropathogenic Escherichia coli(Springer Nature, 2021-04-23) Saxena, Vijay; Gao, Hongyu; Arregui, Samuel; Zollman, Amy; Kamocka, Malgorzata Maria; Xuei, Xiaoling; McGuire, Patrick; Hutchens, Michael; Hato, Takashi; Hains, David S.; Schwaderer, Andrew L.; Pediatrics, School of MedicineKidney intercalated cells are involved in acid-base homeostasis via vacuolar ATPase expression. Here we report six human intercalated cell subtypes, including hybrid principal-intercalated cells identified from single cell transcriptomics. Phagosome maturation is a biological process that increases in biological pathway analysis rank following exposure to uropathogenic Escherichia coli in two of the intercalated cell subtypes. Real time confocal microscopy visualization of murine renal tubules perfused with green fluorescent protein expressing Escherichia coli or pHrodo Green E. coli BioParticles demonstrates that intercalated cells actively phagocytose bacteria then acidify phagolysosomes. Additionally, intercalated cells have increased vacuolar ATPase expression following in vivo experimental UTI. Taken together, intercalated cells exhibit a transcriptional response conducive to the kidney’s defense, engulf bacteria and acidify the internalized bacteria. Intercalated cells represent an epithelial cell with characteristics of professional phagocytes like macrophages.Item Longitudinal SARS-CoV-2 seroconversion and functional heterogeneity in a pediatric dialysis unit(Elsevier, 2021-02) Canas, Jorge J.; Starr, Michelle C.; Arregui, Samuel; Wilson, Amy C.; Carroll, Aaron E.; Saxena, Vijay; Amanat, Fatima; Krammer, Florian; Fill, Jeffrey; Schade, Andrew; Chambers, Antonio; Schneider, Jack; Schwaderer, Andrew L.; Hains, David S.; Pediatrics, School of MedicineItem MAP3K7 is an innate immune regulatory gene with increased expression in human and murine kidney intercalated cells following uropathogenic Escherichia coli exposure(Wiley, 2022) Saxena, Vijay; Arregui, Samuel; Kamocka, Malgorzata Maria; Hains, David S.; Schwaderer, Andrew; Pediatrics, School of MedicineUnderstanding the mechanisms responsible for the kidney's defense against ascending uropathogen is critical to devise novel treatment strategies against increasingly antibiotic resistant uropathogen. Growing body of evidence indicate Intercalated cells of the kidney as the key innate immune epithelial cells against uropathogen. The aim of this study was to find orthologous and differentially expressed bacterial defense genes in human versus murine intercalated cells. We simultaneously analyzed 84 antibacterial genes in intercalated cells enriched from mouse and human kidney samples. Intercalated cell “reporter mice” were exposed to saline versus uropathogenic Escherichia coli (UPEC) transurethrally for 1 h in vivo, and intercalated cells were flow sorted. Human kidney intercalated cells were enriched from kidney biopsy using magnetic‐activated cell sorting and exposed to UPEC in vitro for 1 h. RT2 antibacterial PCR array was performed. Mitogen‐activated protein kinase kinase kinase 7 (MAP3K7) messenger RNA (mRNA) expression increased in intercalated cells of both humans and mice following UPEC exposure. Intercalated cell MAP3K7 protein expression was defined by immunofluorescence and confocal imaging analysis, was consistent with the increased MAP3K7 mRNA expression profiles defined by PCR. The presence of the orthologous innate immune gene MAP3K7/TAK1 suggests that it may be a key regulator of the intercalated cell antibacterial response and demands further investigation of its role in urinary tract infection pathogenesis.Item A Pilot Single Cell Analysis of the Zebrafish Embryo Cellular Responses to Uropathogenic Escherichia coli Infection(Case Western Reserve University, 2022-02-04) Rawson, Ashley; Saxena, Vijay; Gao, Hongyu; Hooks, Jenaya; Xuei, Xiaoling; McGuire, Patrick; Hato, Takashi; Hains, David S.; Anderson, Ryan M.; Schwaderer, Andrew L.; Pediatrics, School of MedicineBackground: Uropathogenic Escherichia coli (UPEC) infections are common and when they disseminate can be of high morbidity. Methods: We studied the effects of UPEC infection using single cell RNA sequencing (scRNAseq) in zebrafish. Bulk RNA sequencing has historically been used to evaluate gene expression patterns, but scRNAseq allows gene expression to be evaluated at the single cell level and is optimal for evaluating heterogeneity within cell types and rare cell types. Zebrafish cohorts were injected with either saline or UPEC, and scRNAseq and canonical pathway analyses were performed. Results: Canonical pathway analysis of scRNAseq data provided key information regarding innate immune pathways in the cells determined to be thymus cells, ionocytes, macrophages/monocytes, and pronephros cells. Pathways activated in thymus cells included interleukin 6 (IL-6) signaling and production of reactive oxygen species. Fc receptor-mediated phagocytosis was a leading canonical pathway in the pronephros and macrophages. Genes that were downregulated in UPEC vs saline exposed embryos involved the cellular response to the Gram-negative endotoxin lipopolysaccharide (LPS) and included Forkhead Box O1a (Foxo1a), Tribbles Pseudokinase 3 (Trib3), Arginase 2 (Arg2) and Polo Like Kinase 3 (Plk3). Conclusions: Because 4-day post fertilization zebrafish embryos only have innate immune systems, the scRNAseq provides insights into pathways and genes that cell types utilize in the bacterial response. Based on our analysis, we have identified genes and pathways that might serve as genetic targets for treatment and further investigation in UPEC infections at the single cell level.