Murine Ribonuclease 6 Limits Bacterial Dissemination during Experimental Urinary Tract Infection.

INTRODUCTION: The ribonuclease (RNase) A superfamily encodes cationic antimicrobial proteins with potent microbicidal activity toward uropathogenic bacteria. Ribonuclease 6 (RNase6) is an evolutionarily conserved, leukocyte-derived antimicrobial peptide with potent microbicidal activity toward uropathogenic Escherichia coli (UPEC), the most common cause of bacterial urinary tract infections (UTIs). In this study, we generated Rnase6-deficient mice to investigate the hypothesis that endogenous RNase 6 limits host susceptibility to UTI. METHODS: We generated a Rnase6EGFP knock-in allele to identify cellular sources of Rnase6 and determine the consequences of homozygous Rnase6 deletion on antimicrobial activity and UTI susceptibility. RESULTS: We identified monocytes and macrophages as the primary cellular sources of Rnase6 in bladders and kidneys of Rnase6EGFP/+ mice. Rnase6 deficiency (i.e., Rnase6EGFP/EGFP) resulted in increased upper urinary tract UPEC burden during experimental UTI, compared to Rnase6+/+ controls. UPEC displayed increased intracellular survival in Rnase6-deficient macrophages. CONCLUSION: Our findings establish that RNase6 prevents pyelonephritis by promoting intracellular UPEC killing in monocytes and macrophages and reinforce the overarching contributions of endogenous antimicrobial RNase A proteins to host UTI defense.

Biomarkers for urinary tract infection: present and future perspectives.

A prompt diagnosis of urinary tract infection (UTI) is necessary to minimize its symptoms and limit sequelae. The current UTI screening by urine test strip analysis and microscopic examination has suboptimal diagnostic accuracy. A definitive diagnosis of UTI by urine culture takes two to three days for the results. These limitations necessitate a need for better biomarkers for the diagnosis and subsequent management of UTI in children. Here, we review the value of currently available UTI biomarkers and highlight the potential of emerging biomarkers that can facilitate a more rapid and accurate UTI diagnosis. Of the newer UTI biomarkers, the most promising are blood procalcitonin (PCT) and urinary neutrophil gelatinase-associated lipocalin (NGAL). PCT can provide diagnostic benefits and should be considered in patients who have a blood test for other reasons. NGAL, which is on the threshold of clinical care, needs more research to address its scope and utilization, including point-of-care application. Employment of these and other biomarkers may ultimately improve UTI diagnosis, guide UTI therapy, reduce antibiotic use, and mitigate UTI complications.

Human Ribonuclease 6 Has a Protective Role during Experimental Urinary Tract Infection.

Mounting evidence suggests that antimicrobial peptides and proteins (AMPs) belonging to the RNase A superfamily have a critical role in defending the bladder and kidney from bacterial infection. RNase 6 has been identified as a potent, leukocyte-derived AMP, but its impact on urinary tract infection (UTI) in vivo has not been demonstrated. To test the functional role of human RNase 6, we generated RNASE6 transgenic mice and studied their susceptibility to experimental UTI. In addition, we generated bone marrow-derived macrophages to study the impact of RNase 6 on antimicrobial activity within a cellular context. When subjected to experimental UTI, RNASE6 transgenic mice developed reduced uropathogenic Escherichia coli (UPEC) burden, mucosal injury, and inflammation compared to non-transgenic controls. Monocytes and macrophages were the predominant cellular sources of RNase 6 during UTI, and RNASE6 transgenic macrophages were more proficient at intracellular UPEC killing than non-transgenic controls. Altogether, our findings indicate a protective role for human RNase 6 during experimental UTI.

Uropathogen and host responses in pyelonephritis.

Urinary tract infections (UTIs) are among the most common bacterial infections seen in clinical practice. The ascent of UTI-causing pathogens to the kidneys results in pyelonephritis, which can trigger kidney injury, scarring and ultimately impair kidney function. Despite sizable efforts to understand how infections develop or are cleared in the bladder, our appreciation of the mechanisms by which infections develop, progress or are eradicated in the kidney is limited. The identification of virulence factors that are produced by uropathogenic Escherichia coli to promote pyelonephritis have begun to fill this knowledge gap, as have insights into the mechanisms by which kidney tubular epithelial cells oppose uropathogenic E. coli infection to prevent or eradicate UTIs. Emerging data also illustrate how specific cellular immune responses eradicate infection whereas other immune cell populations promote kidney injury. Insights into the mechanisms by which uropathogenic E. coli circumvent host immune defences or antibiotic therapy to cause pyelonephritis is paramount to the development of new prevention and treatment strategies to mitigate pyelonephritis and its associated complications.

Neutrophil-Macrophage Imbalance Drives the Development of Renal Scarring during Experimental Pyelonephritis.

BACKGROUND: In children, the acute pyelonephritis that can result from urinary tract infections (UTIs), which commonly ascend from the bladder to the kidney, is a growing concern because it poses a risk of renal scarring and irreversible loss of kidney function. To date, the cellular mechanisms underlying acute pyelonephritis-driven renal scarring remain unknown. METHODS: We used a preclinical model of uropathogenic Escherichia coli-induced acute pyelonephritis to determine the contribution of neutrophils and monocytes to resolution of the condition and the subsequent development of kidney fibrosis. We used cell-specific monoclonal antibodies to eliminate neutrophils, monocytes, or both. Bacterial ascent and the cell dynamics of phagocytic cells were assessed by biophotonic imaging and flow cytometry, respectively. We used quantitative RT-PCR and histopathologic analyses to evaluate inflammation and renal scarring. RESULTS: We found that neutrophils are critical to control bacterial ascent, which is in line with previous studies suggesting a protective role for neutrophils during a UTI, whereas monocyte-derived macrophages orchestrate a strong, but ineffective, inflammatory response against uropathogenic, E. coli-induced, acute pyelonephritis. Experimental neutropenia during acute pyelonephritis resulted in a compensatory increase in the number of monocytes and heightened macrophage-dependent inflammation in the kidney. Exacerbated macrophage-mediated inflammatory responses promoted renal scarring and compromised renal function, as indicated by elevated serum creatinine, BUN, and potassium. CONCLUSIONS: These findings reveal a previously unappreciated outcome for neutrophil-macrophage imbalance in promoting host susceptibility to acute pyelonephritis and the development of permanent renal damage. This suggests targeting dysregulated macrophage responses might be a therapeutic tool to prevent renal scarring during acute pyelonephritis.

Analysis of the Ribonuclease A Superfamily of Antimicrobial Peptides in Patients Undergoing Chronic Peritoneal Dialysis.

Infectious peritonitis is a common complication in patients undergoing chronic peritoneal dialysis (PD), limiting the duration of PD as a modality for renal replacement therapy and increasing patient morbidity and mortality. Antimicrobial peptides (AMPs) serve critical roles in mucosal defense, but their expression and activity during peritonitis are poorly understood. We hypothesized that AMPs belonging to the Ribonuclease (RNase) A Superfamily are present in peritoneal fluid and increase during peritonitis in patients undergoing chronic PD. In the absence of peritonitis, we detected RNase 3, RNase 6, and RNase 7 in cell-free supernatants and viable cells obtained from peritoneal fluid of chronic PD patients. The cellular sources of these RNases were eosinophils (RNase 3), macrophages (RNase 6), and mesothelial cells (RNase 7). During peritonitis, RNase 3 increased 55-fold and RNase 7 levels increased 3-fold on average, whereas RNase 6 levels were unchanged. The areas under the receiver-operating characteristic curves for RNase 3 and RNase 7 were 0.99 (95% confidence interval (CI): 0.96-1.0) and 0.79 (95% CI: 0.64-0.93), respectively, indicating their potential as biomarkers of peritonitis. Discrete omental reservoirs of these RNases were evident in patients with end stage kidney disease prior to PD initiation, and omental RNase 3 reactive cells increased in patients undergoing PD with a history of peritonitis. We propose that constitutive and inducible pools of antimicrobial RNases form a network to shield the peritoneal cavity from microbial invasion in patients undergoing chronic PD.

The clinical diagnosis and management of urinary tract infections in children and adolescents.

Urinary tract infections (UTI) are one of the most common and serious bacterial infections encountered by paediatricians and primary care physicians. Although the diagnosis and management of UTI appear simplistic, they remain among the most contentious issues in paediatrics. In part, UTI controversies stem from the absence of classic clinical symptoms, inappropriate urine specimen collection, modified urinary tract imaging recommendations, and diverse treatment and prevention strategies. Recently published guidelines and large clinical trials have attempted to clarify UTI diagnostic and management strategies. In this manuscript, we review the diagnosis and management of acute and recurrent UTI in the paediatric and adolescent populations.

Insulin and the phosphatidylinositol 3-kinase signaling pathway regulate Ribonuclease 7 expression in the human urinary tract.

Diabetes mellitus is a systemic disease associated with a deficiency of insulin production or action. Diabetic patients have an increased susceptibility to infection with the urinary tract being the most common site. Recent studies suggest that Ribonuclease 7 (RNase 7) is a potent antimicrobial peptide that plays an important role in protecting the urinary tract from bacterial insult. Because the impact of diabetes on RNase 7 expression and function are unknown, we investigated the effects of insulin on RNase 7 using human urine specimens. The urinary RNase 7 concentrations were measured in healthy control patients and insulin-deficient type 1 diabetics before and after starting insulin therapy. Compared with controls, diabetic patients had suppressed urinary RNase 7 concentrations, which increased with insulin. Using primary human urothelial cells, the mechanisms by which insulin stimulates RNase 7 synthesis were next explored. Insulin induced RNase 7 production via the phosphatidylinositide 3-kinase signaling pathway (PI3K/AKT) to shield urothelial cells from uropathogenic E. coli. In contrast, uropathogenic E. coli suppressed PI3K/AKT activity and RNase 7 production. Thus, insulin and PI3K/AKT signaling are essential for RNase 7 expression and increased infection risks in diabetic patients may be secondary to suppressed RNase 7 production. Our data may provide unique insight into novel urinary tract infection therapeutic strategies in at-risk populations.

Expression and Significance of the HIP/PAP and RegIIIγ Antimicrobial Peptides during Mammalian Urinary Tract Infection.

Recent evidence indicates that antimicrobial peptides (AMPs) serve key roles in defending the urinary tract against invading uropathogens. To date, the individual contribution of AMPs to urinary tract host defense is not well defined. In this study, we identified Regenerating islet-derived 3 gamma (RegIIIγ) as the most transcriptionally up-regulated AMP in murine bladder transcriptomes following uropathogenic Escherichia coli (UPEC) infection. We confirmed induction of RegIIIγ mRNA during cystitis and pyelonephritis by quantitative RT-PCR. Immunoblotting demonstrates increased bladder and urinary RegIIIγ protein levels following UPEC infection. Immunostaining localizes RegIIIγ protein to urothelial cells of infected bladders and kidneys. Human patients with UTI have increased urine concentrations of the orthologous Hepatocarcinoma-Intestine-Pancreas / Pancreatitis Associated Protein (HIP/PAP) compared to healthy controls. Recombinant RegIIIγ protein does not demonstrate bactericidal activity toward UPEC in vitro, but does kill Staphylococcus saprophyticus in a dose-dependent manner. Kidney and bladder tissue from RegIIIγ knockout mice and wild-type mice contain comparable bacterial burden following UPEC and Gram-positive UTI. Our results demonstrate that RegIIIγ and HIP/PAP expression is induced during human and murine UTI. However, their specific function in the urinary tract remains uncertain.

Amplifying renal immunity: the role of antimicrobial peptides in pyelonephritis.

Urinary tract infections (UTIs), including pyelonephritis, are among the most common and serious infections encountered in nephrology practice. UTI risk is increased in selected patient populations with renal and urinary tract disorders. As the prevalence of antibiotic-resistant uropathogens increases, novel and alternative treatment options will be needed to reduce UTI-associated morbidity. Discoveries over the past decade demonstrate a fundamental role for the innate immune system in protecting the urothelium from bacterial challenge. Antimicrobial peptides, an integral component of this urothelial innate immune system, demonstrate potent bactericidal activity toward uropathogens and might represent a novel class of UTI therapeutics. The urothelium of the bladder and the renal epithelium secrete antimicrobial peptides into the urinary stream. In the kidney, intercalated cells--a cell-type involved in acid-base homeostasis--have been shown to be an important source of antimicrobial peptides. Intercalated cells have therefore become the focus of new investigations to explore their function during pyelonephritis and their role in maintaining urinary tract sterility. This Review provides an overview of UTI pathogenesis in the upper and lower urinary tract. We describe the role of intercalated cells and the innate immune response in preventing UTI, specifically highlighting the role of antimicrobial peptides in maintaining urinary tract sterility.

Expression and antimicrobial function of beta-defensin 1 in the lower urinary tract.

Beta defensins (BDs) are cationic peptides with antimicrobial activity that defend epithelial surfaces including the skin, gastrointestinal, and respiratory tracts. However, BD expression and function in the urinary tract are incompletely characterized. The purpose of this study was to describe Beta Defensin-1 (BD-1) expression in the lower urinary tract, regulation by cystitis, and antimicrobial activity toward uropathogenic Escherichia coli (UPEC) in vivo. Human DEFB1 and orthologous mouse Defb1 mRNA are detectable in bladder and ureter homogenates, and human BD-1 protein localizes to the urothelium. To determine the relevance of BD-1 to lower urinary tract defense in vivo, we evaluated clearance of UPEC by Defb1 knockout (Defb1(-/-)) mice. At 6, 18, and 48 hours following transurethral UPEC inoculation, no significant differences were observed in bacterial burden in bladders or kidneys of Defb1(-/-) and wild type C57BL/6 mice. In wild type mice, bladder Defb1 mRNA levels decreased as early as two hours post-infection and reached a nadir by six hours. RT-PCR profiling of BDs identified expression of Defb3 and Defb14 mRNA in murine bladder and ureter, which encode for mBD-3 and mBD-14 protein, respectively. MBD-14 protein expression was observed in bladder urothelium following UPEC infection, and both mBD-3 and mBD-14 displayed dose-dependent bactericidal activity toward UPEC in vitro. Thus, whereas mBD-1 deficiency does not alter bladder UPEC burden in vivo, we have identified mBD-3 and mBD-14 as potential mediators of mucosal immunity in the lower urinary tract.

The accuracy and health risks of a voiding cystourethrogram after a febrile urinary tract infection.

OBJECTIVE: Physicians often defer obtaining a voiding cystourethrogram (VCUG) after the diagnosis of urinary tract infection (UTI) due to concerns regarding increased health risks and inflated rates of vesicoureteral reflux (VUR). This study examines the health risks and accuracy of VCUG testing after diagnosis of a febrile UTI. PATIENTS AND METHODS: A retrospective review was conducted of children aged 0-18 years admitted to Nationwide Children's Hospital with a febrile UTI in 1995-2000. Children were divided into two cohorts - those who had a VCUG performed within 1 week of diagnosis (early VCUG cohort) and those who had a VCUG performed more than 1 week after diagnosis (late VCUG cohort). All children were followed for an additional 5 years after hospital discharge. RESULTS: The incidence and severity of VUR were similar in patients that underwent early and late VCUG testing. Patients who underwent early VCUG testing showed no sign of worsening illness after the test was performed. During the 5-year follow up, these patients did not have higher rates of return emergency department visits or hospital readmission compared to those who received late VCUG testing. CONCLUSIONS: The rate of VUR detection does not increase with early VCUG testing. Early VCUG testing does not lead to increased risk of bacterial dissemination or urosepsis.