DEFA1A3 DNA gene-dosage regulates the kidney innate immune response during upper urinary tract infection.

Antimicrobial 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.

Generation of Atp6v1g3-Cre mice for investigation of intercalated cells and the collecting duct.

Kidney intercalated cells (ICs) maintain acid-base homeostasis and recent studies have demonstrated that they function in the kidney's innate defense. To study kidney innate immune function, ICs have been enriched using vacuolar ATPase (V-ATPase) B1 subunit (Atp6v1b1)-Cre (B1-Cre) mice. Although Atp6v1b1 is considered kidney specific, it is expressed in multiple organ systems, both in mice and humans, raising the possibility of off-target effects when using the Cre-lox system. We have recently shown using single-cell RNA sequencing that the gene that codes for the V-ATPase G3 subunit (mouse gene: Atp6v1g3; human gene: ATP6V1G3; protein abbreviation: G3) mRNA is selectively enriched in human kidney ICs. In this study, we generated Atp6v1g3-Cre (G3-Cre) reporter mice using CRISPR/CAS technology and crossed them with Tdtomatoflox/flox mice. The resultant G3-Cre+Tdt+ progeny was evaluated for kidney specificity in multiple tissues and found to be highly specific to kidney cells with minimal or no expression in other organs evaluated compared with B1-Cre mice. Tdt+ cells were flow sorted and were enriched for IC marker genes on RT-PCR analysis. Next, we crossed these mice to ihCD59 mice to generate an IC depletion mouse model (G3-Cre+ihCD59+/+). ICs were depleted in these mice using intermedilysin, which resulted in lower blood pH, suggestive of a distal renal tubular acidosis phenotype. The G3-Cre mice were healthy, bred normally, and produce regular-sized litter. Thus, this new "IC reporter" mice can be a useful tool to study ICs.NEW & NOTEWORTHY This study details the development, validation, and experimental use of a new mouse model to study the collecting duct and intercalated cells. Kidney intercalated cells are a cell type increasingly recognized to be important in several human diseases including kidney infections, acid-base disorders, and acute kidney injury.

Human neutrophil peptides 1-3 protect the murine urinary tract from uropathogenic Escherichia coli challenge.

Antimicrobial 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.

MAP3K7 is an innate immune regulatory gene with increased expression in human and murine kidney intercalated cells following uropathogenic Escherichia coli exposure.

Understanding 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.

Placement on COVID-19 Units Does Not Increase Seroconversion Rate of Pediatric Graduate Medical Residents.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated disease COVID-19 (coronavirus disease 2019) has presented graduate medical education (GME) training programs with a unique set of challenges. One of the most pressing is how should hospital systems that rely on graduate medical residents provide appropriate care for patients while protecting trainees. This question is of particular concern as healthcare workers are at high risk of SARS-CoV-2 exposure. Objective: This cross-sectional study sought to assess the impact of hospital COVID-19 patient placement on pediatric graduate medical residents by comparing rates of SARS-CoV-2 seroconversion rates of residents who worked on designated COVID-19 teams and those who did not. Methods: Forty-four pediatric and medicine-pediatric residents at Riley Children's Hospital (Indianapolis, IN) were tested for SARS-CoV-2 immunoglobulin M (IgM) and IgG seroconversion in May 2020 using enzyme-linked immunosorbent assays (Abnova catalog no. KA5826), 2 months after the first known COVID-19 case in Indiana. These residents were divided into two groups: those residents who worked on designated COVID-19 teams, and those who did not. Groups were compared using χ2 or Fisher exact test for categorical variables, and continuous variables were compared using Student t testing. Results: Forty-four of 104 eligible residents participated in this study. Despite high rates of seroconversion, there was no difference in the risk of SARS-CoV-2 seroconversion between residents who worked on designated COVID-19 teams (26% or 8/31) and those who did not (31% or 4/13). Eleven of 44 residents (25%) tested positive for SARS-CoV-2 IgG, whereas only 5/44 (11.4%) tested positive for SARS-CoV-2 IgM, without a detectable difference between exposure groups. Conclusion: We did not observe a difference in SARS-CoV-2 seroconversion between different exposure groups. These data are consistent with growing evidence supporting the efficacy of personal protective equipment. Further population-based research on the role of children in transmitting the SARS-CoV-2 virus is needed to allow for a more evidence-based approach toward managing the COVID-19 pandemic.

Kidney intercalated cells are phagocytic and acidify internalized uropathogenic Escherichia coli.

Kidney 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.

Assessment of Seroconversion to SARS-CoV-2 in a Cohort of Pediatric Kidney Transplant Recipients.

Background: The occurrence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the associated coronavirus disease 2019 (COVID-19) have profoundly affected adult kidney disease patients. In contrast, pediatric solid organ transplant recipients, including pediatric kidney transplant (KT) recipients, do not seem to be at particularly higher risk for SARS-CoV-2 infection or for severe COVID-19 disease. This patient population might be protected by certain mechanisms, such as the immunosuppressive medications with their anti-inflammatory properties or simply being well-versed in self-protection techniques. Assessing SARS-CoV-2 antibody serologies could potentially help understand why this patient population is apparently spared from severe SARS-CoV-2 clinical courses. Objective: To examine SARS-CoV-2 serologic status in a cohort of pediatric KT recipients. Methods: SARS-CoV-2 anti-spike IgG and IgM antibodies were measured by three different methods in pediatric KT recipients coming for routine clinic visits immediately post-confinement in May-June of 2020. The patients were considered seroconverted if SARS-CoV-2 antibodies were positive by 2/3 methods and weak positive/indeterminate if positive by 1/3. Results: Thirty-one patients were evaluated (about 1/3 of our institution's pediatric KT population). One patient seroconverted, while three were considered weak positive/indeterminate. None were symptomatic and none had nasopharyngeal PCR confirmed SARS-CoV-2 disease. Conclusions: Seroconversion to SARS-CoV-2 was rare in this population and likely reflects the social distancing practiced by these patients. The results will serve as a foundation for a future longitudinal study to evaluate the long-term emergence and persistence of antibodies in this population and may inform studies of response to a future vaccine.

Aptamer based proteomic pilot study reveals a urine signature indicative of pediatric urinary tract infections.

OBJECTIVE: Current urinary tract infection (UTI) diagnostic strategies that rely on leukocyte esterase have limited accuracy. We performed an aptamer-based proteomics pilot study to identify urine protein levels that could differentiate a culture proven UTI from culture negative samples, regardless of pyuria status. METHODS: We analyzed urine from 16 children with UTIs, 8 children with culture negative pyuria and 8 children with negative urine culture and no pyuria. The urine levels of 1,310 proteins were quantified using the Somascan™ platform and normalized to urine creatinine. Machine learning with support vector machine (SVM)-based feature selection was performed to determine the combination of urine biomarkers that optimized diagnostic accuracy. RESULTS: Eight candidate urine protein biomarkers met filtering criteria. B-cell lymphoma protein, C-X-C motif chemokine 6, C-X-C motif chemokine 13, cathepsin S, heat shock 70kDA protein 1A, mitogen activated protein kinase, protein E7 HPV18 and transgelin. AUCs ranged from 0.91 to 0.95. The best prediction was achieved by the SVMs with radial basis function kernel. CONCLUSIONS: Biomarkers panel can be identified by the emerging technologies of aptamer-based proteomics and machine learning that offer the potential to increase UTI diagnostic accuracy, thereby limiting unneeded antibiotics.

Developmental loss, but not pharmacological suppression, of renal carbonic anhydrase 2 results in pyelonephritis susceptibility.

Carbonic anhydrase II knockout (Car2-/-) mice have depleted numbers of renal intercalated cells, which are increasingly recognized to be innate immune effectors. We compared pyelonephritis susceptibility following reciprocal renal transplantations between Car2-/- and wild-type mice. We examined the effect of pharmacological CA suppression using acetazolamide in an experimental murine model of urinary tract infection. Car2-/- versus wild-type mice were compared for differences in renal innate immunity. In our transplant scheme, mice lacking CA-II in the kidney had increased pyelonephritis risk. Mice treated with acetazolamide had lower kidney bacterial burdens at 6 h postinfection, which appeared to be due to tubular flow from diuresis because comparable results were obtained when furosemide was substituted for acetazolamide. Isolated Car2-/- kidney cells enriched for intercalated cells demonstrated altered intercalated cell innate immune gene expression, notably increased calgizzarin and insulin receptor expression. Intercalated cell number and function along with renal tubular flow are determinants of pyelonephritis risk.