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.

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.

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.

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.

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.

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.

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.

Physiological response of crossbred sheep under nutritional scarcity.

Nutritional scarcity during summer and winter is a major constraint for sheep rearing in the semi-arid tropical region. In this region, a new crossbred sheep has been developed by the crossing of a native sheep breed Malpura with a breed of hot-humid coastal region breed Garole. A study was conducted for 9 weeks to assess the physiological response of crossbred (Garole x Malpura x Malpura, GMM) rams under nutritional scarcity in the semi-arid tropical region. Eighteen adult crossbred rams were randomly allocated into three groups of six animals each. The groups are G1 (control, ad libitum feeding), G2 (20% less than ad libitum feeding), and G3 (30% less than ad libitum feeding). The animals were stall-fed with a diet consisting of 70% roughage (Cenchrus ciliaris hay) and 30% concentrate feed. Body weight gain, average daily gain, and feed intake were significantly (P < 0.05) lower in G2 and G3 as compared to G1 rams. Water intake was significantly (P < 0.05) higher in G2 and G3. Physiological responses, blood biochemical and endocrine profile did not differ between the groups. The results indicate that the crossbred rams evolved through the crossing of the hot semi-arid tropical region breed with hot humid coastal region breed are able to maintain their body homeostasis despite nutritional scarcity although their body weight decreased.

Cell-specific qRT-PCR of renal epithelial cells reveals a novel innate immune signature in murine collecting duct.

The urinary tract is usually culture negative despite its close proximity to microbial flora. The precise mechanism by which the kidneys and urinary tract defends against infection is not well understood. The initial kidney cells to encounter ascending pathogens are the collecting tubule cells that consist of principal cells (PCs) that express aquaporin 2 (AQP2) and intercalated cells (ICs) that express vacuolar H+-ATPase (V-ATPase, B1 subunit). We have previously shown that ICs are involved with the human renal innate immune defense. Here we generated two reporter mice, VATPase B1-cre+tdT+ mice to fluorescently label ICs and AQP2-cre+tdT+ mice to fluorescently label PCs, and then performed flow sorting to enrich PCs and ICs for analysis. Isolated ICs and PCs along with proximal tubular cells were used to measure antimicrobial peptide (AMP) mRNA expression. ICs and PCs were significantly enriched for AMPs. Isolated ICs responded to uropathogenic Escherichia coli (UPEC) challenge in vitro and had higher RNase4 gene expression than control while both ICs and PCs responded to UPEC challenge in vivo by upregulating Defb1 mRNA expression. To our knowledge, this is the first report of isolating murine collecting tubule cells and performing targeted analysis for multiple classes of AMPs.

The gut-liver axis: impact of a mouse model of small-bowel bacterial overgrowth.

BACKGROUND: The mechanisms by which intestinal bacteria impact liver diseases remain poorly understood. The aim of this study was to develop a mouse model of small-bowel bacterial overgrowth and to determine its impact on hepatobiliary injury. MATERIALS AND METHODS: A jejunal self-filling blind loop (SFBL) was created in C57BL/6 mice. Three weeks after surgery, the mice were euthanized, and bacterial cultures of luminal content of the loop and extraintestinal tissues were performed. Liver and jejunum were collected for histological grading of inflammation and injury. Serum liver biochemistry assays were performed. Hepatobiliary transporter mRNA expression in liver was measured by quantitative real-time polymerase chain reaction. Bile and blood were collected for measurement of total bile acids, phospholipid, and cholesterol. Mice undergoing jejunal transection and reanastomosis and laparotomy only served as control groups. RESULTS: SFBL induced a dramatic increase in intraluminal bacterial counts, mesenteric lymph node bacterial translocation, and evidence of jejunal and hepatobiliary injury. Significant reductions in hepatic expression of hepatobiliary transporters involved in biliary canalicular export and basolateral uptake were observed in SFBL mice. SFBL resulted in a significant increase in biliary total bile acid concentration, decreases in bile phospholipid and cholesterol output, and an increase in the bile acid/phospholipid ratio. CONCLUSIONS: We have developed a reproducible mouse model of small-bowel bacterial overgrowth with evidence of liver inflammation, altered hepatobiliary transporter expression, and alterations in bile composition. This model may help to elucidate the mechanisms by which gut-derived bacterial factors impact the liver and contribute to the exacerbation of liver diseases and biliary injury.

Renal Calcium Oxalate Deposits Induce a Pro-Atherosclerotic and Pro-Osteoporotic Response in Mice.

Urinary stone disease (USD) is increasing in adult and pediatric populations. Adult and pediatric studies have demonstrated decreased bone mineral density and increased fracture rates. USD has also been independently linked to increased rates of myocardial infarction and cerebral vascular accidents. Although USD is a multisystem disorder involving the kidneys, bone, and vasculature, the molecular mechanisms linking these three organs remain unknown. Calcium oxalate nephropathy was induced in C57BL/6J mice with intra-peritoneal (ip) injection of sodium glyoxolate. Half of each kidney underwent Pizzalato staining and half was snap frozen for RNA extraction. RT2 Profiler Mouse Atherosclerosis, Osteoporosis, and Calcium Signaling PCR Arrays (Qiagen) were performed. Only results that passed quality checks in PCR array reproducibility and genomic DNA contamination were included. Genes had to show at least fourfold differential expression and P < 0.01 to be considered significant. Atherosclerosis array showed upregulation of 19 genes by fourfold, 10 of which were ≥10-fold. All 19 had P ≤ 0.002. The Osteoporosis array showed fourfold upregulation of 10 genes, five showed >10-fold increase. All 10 have P ≤ 0.003. The calcium signaling array showed significant fourfold upregulation of 10 genes, four of which were ≥10-fold. All 10 have P ≤ 0.03. We have demonstrated that calcium oxalate nephropathy can induce upregulation of atherosclerotic, metabolic bone, and calcium homeostasis genes in a murine model. This may be and initial step in identifying the molecular mechanisms linking stone, bone, and cardiovascular disease. J. Cell. Biochem. 118: 2744-2751, 2017. © 2017 Wiley Periodicals, Inc.

Polymorphisms in α-Defensin-Encoding DEFA1A3 Associate with Urinary Tract Infection Risk in Children with Vesicoureteral Reflux.

The contribution of genetic variation to urinary tract infection (UTI) risk in children with vesicoureteral reflux is largely unknown. The innate immune system, which includes antimicrobial peptides, such as the α-defensins, encoded by DEFA1A3, is important in preventing UTIs but has not been investigated in the vesicoureteral reflux population. We used quantitative real-time PCR to determine DEFA1A3 DNA copy numbers in 298 individuals with confirmed UTIs and vesicoureteral reflux from the Randomized Intervention for Children with Vesicoureteral Reflux (RIVUR) Study and 295 controls, and we correlated copy numbers with outcomes. Outcomes studied included reflux grade, UTIs during the study on placebo or antibiotics, bowel and bladder dysfunction, and renal scarring. Overall, 29% of patients and 16% of controls had less than or equal to five copies of DEFA1A3 (odds ratio, 2.09; 95% confidence interval, 1.40 to 3.11; P<0.001). For each additional copy of DEFA1A3, the odds of recurrent UTI in patients receiving antibiotic prophylaxis decreased by 47% when adjusting for vesicoureteral reflux grade and bowel and bladder dysfunction. In patients receiving placebo, DEFA1A3 copy number did not associate with risk of recurrent UTI. Notably, we found that DEFA1A3 is expressed in renal epithelium and not restricted to myeloid-derived cells, such as neutrophils. In conclusion, low DEFA1A3 copy number associated with recurrent UTIs in subjects in the RIVUR Study randomized to prophylactic antibiotics, providing evidence that copy number polymorphisms in an antimicrobial peptide associate with UTI risk.

Molecular characterization of GPR50 gene and study of its comparative genetic variability in sheep breeds adapted to different thermo-contrasting climatic regimens.

GPR50, formerly known as a melatonin-related receptor, is one of the three subtypes of melatonin receptor subfamily, together with MTNR1A and MTNR1B. GPR50, despite its high identity with the melatonin receptor family, does not bind melatonin and is considered to be an ortholog of MTNR1C in mammals. GPR50-expressing cells have been found in the dorsomedial nucleus of the hypothalamus, the periventricular nucleus, and the median eminence. Genetic and functional evidence have been recently investigated linking GPR50 to adaptive thermogenesis and torpor, but still, it is an orphan receptor and is yet to be studied conclusively. The aims of the study were to characterize the GPR50 gene of sheep and to study the sequence variability of the gene in Indian sheep breeds of two different thermo-varied agroclimatic conditions. Genomic DNA isolation was done and a 791-bp sequence was amplified using self-designed primers and SNP profiling done out of samples of all the breeds to study the relative frequency of SNPs in each of the breed. Five important non-synonymous mutations were observed in the various breeds studied. T698G, G1097A, G1270A, G1318A, and C1334G lead to the following substitution: valine by glycine, arginine by glutamine, threonine by alanine, isoleucine by valine, and serine by cytosine, respectively. Two synonymous mutations (T663G and C888T) were also observed in some of the studied breeds. G1270A and C888T were the most prevalent SNPs observed in nearly all of the breeds. C888T SNPs were observed in higher prevalence in Chokla, Marwari, and Magra in comparison to Gaddi and Bharat Merino. A PolyPhen-2 analysis, which is used to assess the potential damaging nature of an SNP, revealed that mutation T698G and G1270A were benign while G1097A, G1318A, and C1334G were damaging with a score of 0.987, 0.993, and 0.739, respectively. A 3-D homology model of the protein was prepared using c4zwjA (UniProt sequence ID) as a template using the online version of Phyre2 protein modeling software. The structure demonstrated closed similarity with other G-coupled receptor and it had a 45 % α-helical content. G1270A and C888T may be taken up for SNP correlation in a larger population study for their association with heat stress protection.

Effect of high ambient temperature on behavior of sheep under semi-arid tropical environment.

High environmental temperature is a major constraint in sheep production under semi-arid tropical environment. Behavior is the earliest indicator of animal's adaptation and responses to the environmental alteration. Therefore, the objective of this study was to assess the effects of high ambient temperature on the behavior of sheep under a semi-arid tropical environment. The experiment was conducted for 6 weeks on 16 Malpura cross (Garole × Malpura × Malpura (GMM)) rams. The rams were divided equally into two groups, designated as C and T. The rams of C were kept in comfortable environmental conditions served as control. The rams of T were exposed to a different temperature at different hours of the day in a climatic chamber, to simulate a high environmental temperature of summer in semi-arid tropic. The behavioral observations were taken by direct instantaneous observation at 15-min intervals for each animal individually. The feeding, ruminating, standing, and lying behaviors were recorded twice a week from morning (0800 hours) to afternoon (1700 hours) for 6 weeks. Exposure of rams to high temperature (T) significantly (P < 0.05) decreased the proportion of time spent in feeding during the observation period in most of the hours of the day as compared to the C. The proportion of time spent in rumination and lying was significantly (P < 0.05) lower in the T group compared to the C. The animals of T spent significantly (P < 0.05) more time in rumination in standing position as compared to the C. The overall proportion of time spent in standing, panting in each hour, and total panting time was significantly (P < 0.05) higher in the T as compared to the C. The result of the study indicates that the exposure of sheep to high ambient temperature severely modulates the behavior of sheep which is directed to circumvent the effect of the stressor.

Effect of thermal exposure on physiological adaptability and seminal attributes of rams under semi-arid environment.

Thermal stress in hot semi-arid environment is a major limitation of sheep production in tropical and sub-tropical climatic condition. The animals tend to maintain homeostasis through physiological adjustments in a hot environment (maximum temperature reaches up to 47.5°C). Therefore, the present study was carried out to assess the effect of thermal exposure on physiological adaptability and seminal attributes of rams under semi-arid environment. The experiment was conducted for eight weeks involving sixteen Malpura crossbred rams (GMM: Garole X Malpura X Malpura). The rams were divided equally into two groups, designated as G1 and G2, respectively. The rams in G1 (Control) group were kept in a sheep shed under naturally prevailing environment without artificial manipulation of ambient temperature (Temperature 30.48±0.38°C; Relative Humidity 28.59±1.15%). The rams of G2 group were exposed to different temperature at different hours of the day (38°C at 1000-1100h; 40°C at 1100-1200h; 42°C at 12:00-1300h; 43°C at 1300-1400h; 44°C at 1400-1500h and 42°C at 1500-1600h) in a climatic chamber for thermal exposure. Physiological responses, blood biochemical profile, blood endocrine profile, sexual behavior and seminal attributes were measured for both the groups. Thermal exposure significantly (P<0.05) increased the water intake; respiration rate, rectal temperature and skin temperature at afternoon in rams. Exposure of rams to thermal stress (G2) significantly (P<0.05) increased cortisol level and decreased tri-ido-thyronine level. The latency period after the first ejaculation, decreased significantly (P<0.05) in G2. The percentage of rapid motile sperm, linearity and average path velocity of sperm were also altered significantly (P<0.05) in thermal exposed rams as compared to control. However, comparable feed intake, body weight, and major blood biochemical parameters, as well as acceptable semen quality attributes of all the rams indicated that the Fec B gene introgressed Malpura cross rams adapted to the thermal exposure under semi-arid tropical climate.

Pediatric Origins of Nephrolithiasis-Associated Atherosclerosis.

OBJECTIVES: To determine if nephrolithiasis-associated atherosclerosis has pediatric origins and to consider possible association between kidney stones and atherosclerosis-related proteins. STUDY DESIGN: We matched children aged 12-17 years with kidney stones and without kidney stones. Carotid artery intima-media thickness (cIMT) was measured by ultrasound. Participants' urine was investigated by enzyme-linked immunosorbent assay for the atherosclerosis-related proteins fibronectin 1, macrophage scavenger receptor 1, osteopontin, and vascular cell adhesion molecule 1 levels, and normalized to urine creatinine levels. RESULTS: Subjects with nephrolithiasis had higher cIMT in the right common carotid artery and overall mean measurement. Urine osteopontin and fibronectin 1 were significant predictors of cIMT. CONCLUSIONS: We have provided initial preliminary evidence that nephrolithiasis-associated atherosclerosis has pediatric origins and performed studies that begin to identify potential reasons for the association of nephrolithiasis and vascular disease.

Study of conformational properties of solid phase synthesized ovine kisspeptin-14 using Circular Dichroism spectroscopy.

Kisspeptins are peptide products of Kiss-1 gene and have been substantially associated with the initiation of puberty by virtue of their ability to cause pulsatile release of GnRH. Kisspeptin consists of 54 amino acids domain called metastin and its biological activity may be localized to the C-terminal (C-10, C-13, and C-14) segments. Kisspeptin binds to its cognate receptor GPR54 in the hypothalamic neurons and it is a G-coupled membrane receptor. This study is an attempt to understand the tentative conformation of the peptides in its native membrane mimicking environment. A 14 amino-acid derivative of kisspeptin (Asp-58-Val59-Ser60-Ala61-Tyr62-Asn63-Trp64-Asn65-Ser66-Phe67-Gly68-Leu69-Arg70-Tyr71NH2) was synthesized by f-moc (9-fluorenyl methoxy carbonyl) solid phase synthesis strategy. The synthetic peptide was cleaved and purified by Reverse phase-HPLC. CD spectroscopic analysis of secondary structure of the peptide revealed random coil disordered conformation in the aqueous environment. However, the peptide adopted more ordered ß-sheet conformation in the solvents such as TFE and HFIP.

Carbonic anhydrase 2 deficiency leads to increased pyelonephritis susceptibility.

Carbonic anhydrase 2 regulates acid-base homeostasis, and recent findings have indicated a correlation between cellular control of acid-base status and the innate defense of the kidney. Mice deficient in carbonic anhydrase 2 (Car2(-/-) mice) have metabolic acidosis, impaired urine acidification, and are deficient in normal intercalated cells. The objective of the present study was to evaluate the biological consequences of carbonic anhydrase 2 deficiency in a murine model of pyelonephritis. Infection susceptibility and transcription of bacterial response components in Car2(-/-) mice were compared with wild-type littermate controls. Car2(-/-) mice had increased kidney bacterial burdens along with decreased renal bacterial clearance after inoculation compared with wild-type mice. Standardization of the urine pH and serum HCO(3)(-) levels did not substantially alter kidney infection susceptibility between wild-type and Car2(-/-) mice; thus, factors other than acid-base status are responsible. Car2(-/-) mice had significantly increased neutrophil-gelatinase-associated lipocalin mRNA and protein and expression at baseline and a marked decreased ability to upregulate key bacterial response genes during pyelonephritis. Our findings provide in vivo evidence that supports a role for carbonic anhydrase 2 and intercalated cells in promoting renal bacterial clearance. Decreased carbonic anhydrase expression results in increased antimicrobial peptide production by cells other than renal intercalated cells, which is not sufficient to prevent infection after a bacterial challenge.

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.