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.

Identification of putative antiviral bioactive compounds derived from family Asteraceae: An in silico approach.

This computational study investigates 21 bioactive compounds from the Asteraceae family as potential inhibitors targeting the Spike protein (S protein) of SARS-CoV-2. Employing in silico methods and simulations, particularly CDOCKER and MM-GBSA, the study identifies two standout compounds, pterodontic acid and cichoric acid, demonstrating robust binding affinities (-46.1973 and -39.4265 kcal/mol) against the S protein. Comparative analysis with Favipiravir underscores their potential as promising inhibitors. Remarkably, these bioactives exhibit favorable ADMET properties, suggesting safety and efficacy. Molecular dynamics simulations validate their stability and interactions, signifying their potential as effective SARS-CoV-2 inhibitors.

Intercalated cell function, kidney innate immunity, and urinary tract infections.

Intercalated cells (ICs) in the kidney collecting duct have a versatile role in acid-base and electrolyte regulation along with the host immune defense. Located in the terminal kidney tubule segment, ICs are among the first kidney cells to encounter bacteria when bacteria ascend from the bladder into the kidney. ICs have developed several mechanisms to combat bacterial infections of the kidneys. For example, ICs produce antimicrobial peptides (AMPs), which have direct bactericidal activity, and in many cases are upregulated in response to infections. Some AMP genes with IC-specific kidney expression are multiallelic, and having more copies of the gene confers increased resistance to bacterial infections of the kidney and urinary tract. Similarly, studies in human children demonstrate that those with history of UTIs are more likely to have single-nucleotide polymorphisms in IC-expressed AMP genes that impair the AMP's bactericidal activity. In murine models, depleted or impaired ICs result in decreased clearance of bacterial load following transurethral challenge with uropathogenic E. coli. A 2021 study demonstrated that ICs even act as phagocytes and acidify bacteria within phagolysosomes. Several immune signaling pathways have been identified in ICs which may represent future therapeutic targets in managing kidney infections or inflammation. This review's objective is to highlight IC structure and function with an emphasis on current knowledge of IC's diverse innate immune capabilities.

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.

Dietary n-3 PUFA augments pre-ovulatory follicle turnover and prolificacy in well-fed ewes.

The present study evaluated the effect of dietary supplementation with n-3 polyunsaturated fatty acids (PUFA) on preovulatory follicle (POF) turnover, prolificacy, and endocrine and metabolic milieu in Malpura sheep. Fifty cyclic ewes with 3-3.5 body condition scores on a five-point scale were allocated equally to two groups (n = 25) following estrus synchronization and were supplemented with 0.6 mL/kg body weight of n-3 PUFA-rich fish oil (FO) or palm oil (PO) as control, for 60 d following an acclimatization period of 7 d. All ewes were mated with sexually active rams at the end of the supplementation period. On ultrasonographic ovarian scanning at the last fourth estrus, the mean number of POFs was 77.8% greater (P < 0.01) in FO ewes than in the PO ewes. The proportion of ewes with multiple ovulations two months after the beginning of supplementation was 56% in the FO group as compared to 8% in the PO group. The number of fetuses was 46% higher (P < 0.01) in the FO than in the PO ewes at d 45 of gestation. At lambing, the twinning percent in the FO ewes was three times greater than in the PO ewes (27.3 vs. 9.1%). Plasma cholesterol, estradiol, and insulin concentrations were lower (P < 0.01) in ewes fed with FO than those offered PO group at the end of the feeding period. It was concluded that the dietary supplementation of n-3 PUFA-rich FO in well-fed Malpura ewes improved the number of follicles and ovulation rate which led to an increased prolificacy, accompanied by a reduction of plasma cholesterols, estradiol, and insulin.

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.

A Pilot Single Cell Analysis of the Zebrafish Embryo Cellular Responses to Uropathogenic Escherichia coli Infection.

BACKGROUND: 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.

De novo pathway is an active metabolic pathway of cysteine synthesis in Haemonchus contortus.

Haemonchus contortus, commonly known as Barber's pole worm, is an economically important gastrointestinal nematode of sheep and goats especially in tropical and sub-tropical regions of the world. Cysteine synthesis is a very important metabolic pathway for the parasite, however the functional aspects of cysteine synthesis in parasite are largely unknown. The key question which we have investigated in the study is; whether the parasite uses a de novo pathway of cysteine synthesis, which is unknown in multicellular organisms of the animal kingdom and known to be absent in mammals. Directional cloning of the cysteine synthase (CS) gene was done in pET303 champion vector using restriction sites XbaI and XhoI. The CS gene of the H.contortus was closely related to CS-A protein of Oesophagostomum dentatum and a hypothetical protein of Ancylostoma ceylanicum. Recombinant protein of the H contortus CS (rHC-CS) gene was expressed using pET303 vector in pLysS BL21 strain of E.coli and subsequently purified by Ni-NTA affinity chromatography. Western blot using anti-His tag antibody confirmed the presence of rHC-CS. Biochemical assay, FTIR and enzyme kinetics studies revealed that rHC-CS used O-acetyl serine as substrate to produce cysteine using de novo pathway and CS activity was also confirmed with the homogenate of H.contortus. Upregulation of CS transcripts in the adult and its downregulation in the L3 larval stage suggests that de novo pathway contributes to the cysteine requirement of mature H.contortus. It is concluded that de novo pathway is an active metabolic pathway in H.contortus.

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.

Effect of nutritional stress on physiological parameters and seminal attributes of native-crossbred ram in semi-arid tropics.

A prolific three-breed (Malpura, Patanwadi, and Garole) cross Avishaan sheep has been developed in the semi-arid zone to improve farmer's income. Nutritional scarcity is a major limitation in animal husbandry during the dearth period of semi-arid tropics. Therefore, before the inaugural launch of the breed into the field, a study was designed to evaluate the effect of nutritional stress on physiological parameters and seminal attributes of native-crossbred rams in semi-arid tropics. Thus, 16 native adapted (Malpura) and 16 native-crossbred rams were equally distributed into four groups, namely, native control (G1), native nutritional stress (G2), native-crossbred control (G3), and native-crossbred nutritional stress (G4). Both the control groups (G1 and G3) were kept on their maintenance requirement as per their body weight, whereas the nutritional stress groups (G2 and G4) were provided 30% less than their maintenance requirement. The body weight of G4 decline (P<0.05) as compared to their initial weight. The plasma glucose level of G2 and G4 reduced (P<0.05) in comparison with G1 and G3, respectively. The total motile sperm percentage, rapid motile sperm percentage, and sperm viability decrease significantly (P<0.05) within the acceptable limit in native-crossbred rams (G4) under nutritional scarcity. However, the similar blood biochemical along with acceptable seminal attributes of all the rams reflected that native-crossbred rams can cope with the nutritional scarcity in semi-arid tropics and have the potential to contribute to the sustainable small ruminant production system for livelihood security in this region.

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.

DCHS1 DNA copy number loss associated with pediatric urinary tract infection risk.

Urinary 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.

Publisher Correction: Whole Transcriptome Analysis of Renal Intercalated Cells Predicts Lipopolysaccharide Mediated Inhibition of Retinoid X Receptor alpha Function.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Adolescents with urinary stones have elevated urine levels of inflammatory mediators.

Urinary stones 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 cardiovascular disease. These aforementioned stone-associated conditions may have pediatric origins. To compare urine inflammatory markers in otherwise healthy stone forming children versus matched controls. 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. Macrophage inflammatory protein 1ß 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. This study indicates that urine levels of cytokines involved in chronic inflammation and fibrosis are elevated in urinary stone formers as early as adolescence. Because stone formers are at risk for chronic kidney disease, macrophage inflammatory protein 1ß and interleukin 13 represent investigative targets.

In silico homology modelling and prediction of novel epitopic peptides from P24 protein of Haemonchus contortus.

Haemonchus contortus (HC) causes Haemonchosis in sheep and goats, with high mortality and morbidity due to lack of effective vaccine and increasing resistance to anthelmintic drugs. The present study was aimed at developing the 3D model of HCP24 protein and to identify the candidate epitopic peptides for effective humoral and cell-mediated immune-response. The HCP24 protein was homology modelled using the Swiss server and developed model was validated by ERRAT, VERIFY3D, PROQ, RAMPAGE and PROCHECK servers. Linear and prominent antigenic epitopes were predicted by SVMTrip and Immuno-medicine group tool. Conformational B-cell epitopes were predicted by Ellipro. MHC-I and MHC-II binding peptides were predicted by MHCPRED2, MHC2PRED and Propred I server. Proteosomal cleavage sites were predicted by Netchop server, to assess the stability of peptides. Reverse and three frame translation was done by EMBOSS tool. Bepipred and IEDB analysis also confirmed that both the predicted peptides (pep-1 and pep-2) were important antigenic region but pep-1 should have better hydrophobicity and stability. The degree of confidence achieved on scientific validation of the generated 3D model of the protein allows us to prescribe its use for research purpose. We could determine the peptide Pep-1(EDCKCTNCVCSRDEAL) should be a conformational B cell epitope with high antigenic potential and should demonstrate good binding affinity with host MHC-II and MHC-I alleles as well as stability inside host. Thus, it could be an ideal vaccine candidate for developing sub-unit vaccine against the parasite and should be assessed for protective immune response by in vitro and in-vivo studies.

Identification and characterization of an M cell marker in nasopharynx- and oropharynx-associated lymphoid tissue of sheep.

M cells play a pivotal role in the induction of immune responses within the mucosa-associated lymphoid tissues. M cells exist principally in the follicle-associated epithelium (FAE) of the isolated solitary lymphoid follicles as well as in the lymphoid follicles of nasopharynx-associated lymphoid tissue and gut associated lymphoid tissue (GALT). Through lymphatic cannulation it is possible to investigate local immune responses induced following nasal Ag delivery in sheep. Hence, identifying sheep M cell markers would allow the targeting of M cells to offset the problem of trans-epithelial Ag delivery associated with inducing mucosal immunity. Sheep cDNA from the tonsils of the oropharynx and nasopharynx was PCR amplified using Glycoprotein-2 (GP2)-specific primers and expressed as a poly-His-tagged recombinant sheep GP2 (56 kDa) in HEK293 cells. The recombinant GP2 protein was purified using Ni-NTA affinity chromatography and polyclonal serum against the protein was raised in rats. The antiserum recognized the recombinant sheep GP2 and purified rat IgG against GP2 stained M cells in sections of sheep tonsils from nasopharynx and oropharynx. M cells were found to be present in epithelium of the palatine tonsils (oropharynx), pharyngeal tonsils as well as tubal tonsils (nasopharynx). They were also present in the FAE of the scattered lymphoid follicles over the base of the nasopharynx. Thus, GP2 has been identified to be an important M cell marker of nasopharynx and oropharynx-associated lymphoid tissues in sheep.