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Antimicrobial peptides and proteins (AMPs) constitute ancient host defence mechanisms to preserve tissue sterility and protect the host from infectious diseases. Currently, AMPs are awakening the interest of medical researchers due to their potential to become novel weapons to target multi‐drug resistant (MDR) pathogens and thereby overcome the limitations of traditional antibiotics. Among AMPs, human RNases belonging to the RNase A superfamily stand out as promising agents for therapeutic uses given their high antimicrobial activity, wide spectrum against multiple pathogens and low toxicity. However, a better understanding of how human RNases perform their antimicrobial actions in tissues is necessary to develop novel therapies. Mouse infectious disease models can be extremely useful to study the function of AMPs in vivo and have already provided valuable knowledge about RNase role in tissues such as the intestine and urinary tract. Therefore, it is necessary to understand the genetic and functional divergences that exist between human and mouse RNases to design experiments that are poised for clinical translation. The aim of this review is to present the similarities and differences between human and mouse RNases at genomic, structural and functional levels as a guide for future scientists exploring the roles of RNases in host defence.

The lower urinary tract is routinely exposed to microbes residing in the gastrointestinal tract, yet the urothelium resists invasive infections by gut microorganisms. This infection resistance is attributed to innate defenses in the bladder urothelium, kidney epithelium, and resident or circulating immune cells. In recent years, surmounting evidence suggests that these cell types produce and secrete soluble host defense peptides, including members of the Ribonuclease (RNase) A Superfamily, to combat invasive bacterial challenge. While some of these peptides, including RNase 4 and RNase 7, are abundantly produced by epithelial cells, the expression of others, like RNase 3 and RNase 6, increase at infection sites with immune cell recruitment. The objective of this mini-review is to highlight recent evidence showing the biological importance and responses of RNase A Superfamily members to infection in the kidney and bladder.

Key Points The innate immune system is essential for preventing urinary tract infection (UTI) and limiting the spread of infection Urinary antimicrobial peptides exhibit bactericidal and bacteriostatic activity toward uropathogenic bacteria Intercalated cells are the major renal source of antimicrobial peptides Antimicrobial peptides possess immunomodulatory activity in addition to antimicrobial activity Urinary antimicrobial peptides have diagnostic and therapeutic potential for patients with UTIs Urinary tract infections (UTIs), including pyelonephritis, are among the most common and serious infections encountered by nephrologists. Emerging evidence suggests a fundamental role for the innate immune system in protecting the urothelium from bacterial challenge. This Review provides an overview of UTI pathogenesis in the upper and lower urinary tract. The authors describe the role of intercalated cells and the innate immune response in preventing UTI, focusing on the role of antimicrobial peptides in maintaining urinary tract sterility. 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.

Urinary tract obstruction (UTO) is a common cause of kidney injury that can result in chronic kidney disease and end-stage renal disease. Heterogeneity in the extent of obstructive renal damage in humans with UTO implies the existence of unknown mechanisms that protect against or accelerate kidney injury. Prior studies show that congenital and acquired UTO initiate a conserved, protective program of renal urothelium remodeling that culminates in expansion of uroplakin (UPK) + cells to promote renal structural integrity. However, the cellular and molecular mechanisms that regulate UPK expression in the renal urothelium are unknown. Peroxisome proliferator-activated receptor γ (PPARγ) drives urothelial differentiation and UPK expression in other tissues but has not been investigated in the renal urothelium. Here we demonstrate that activation of PPARγ in UPK + cells is critical for UTO-induced renal urothelium remodeling. Conditional deletion of Pparg perturbs UPK expression and accelerates parenchymal thinning during UTO, while conditional activation of PPARγ increases UPK expression and results in parenchymal preservation. This study underscores the significance of renal urothelium during UTO and shows that UTO-induced renal urothelial remodeling is achieved through activation of PPARγ. These findings form the foundation for future studies that will determine the therapeutic utility of PPARγ agonists during congenital and acquired UTO. Renal urothelium remodeling protects against kidney injury Urinary tract obstruction (UTO) is a major cause of kidney injury. Emerging evidence highlights the role of uroplakin (UPK) + cells in protecting the kidney during UTO, but the regulatory mechanisms for the UPK expression were unclear. Using bioinformatics, researchers identified peroxisome proliferator-activated receptor γ (PPARγ) as a key upstream regulator, which they validated through conditional genetic manipulation of Pparg during UTO. Deleting PPARγ reduced the UPK expression and accelerated renal thinning, while activating it preserved renal parenchyma. These findings suggest that PPARγ pathways are crucial for protecting the renal urothelium during UTO, providing a potential therapeutic target with PPARγ agonists. The study also reveals significant differences between the upper tract and bladder urothelium, warranting further investigation. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.

The role of calcium oxalate crystals and deposits in UTI pathogenesis has not been established. The objectives of this study were to identify bacteria present in pediatric urolithiasis and, using in vitro and in vivo models, to determine the relevance of calcium oxalate deposits during experimental pyelonephritis. Pediatric kidney stones and urine were collected and both cultured and sequenced for bacteria. Bacterial adhesion to calcium oxalate was compared. Murine kidney calcium oxalate deposits were induced by intraperitoneal glyoxalate injection and kidneys were transurethrally inoculated with uropathogenic Escherichia coli to induce pyelonephritis. E. coli of the family Enterobacteriaceae was identified in patients by calcium oxalate stone culture. Additionally Enterobacteriaceae DNA was sequenced from multiple calcium oxalate kidney stones. E. coli selectively aggregated on and around calcium oxalate monohydrate crystals. Mice inoculated with glyoxalate and uropathogenic E. coli had higher bacterial burdens, increased kidney calcium oxalate deposits and an increased kidney innate immune response compared to mice with only calcium oxalate deposits or only pyelonephritis. In a murine model, the presence of calcium oxalate deposits increases pyelonephritis risk, likely due to preferential aggregation of bacteria on and around calcium oxalate crystals. When both calcium oxalate deposits and uropathogenic bacteria were present, calcium oxalate deposit number increased along with renal gene transcription of inner stone core matrix proteins increased. Therefore renal calcium oxalate deposits may be a modifiable risk factor for infections of the kidney and urinary tract. Furthermore, bacteria may be present in calcium oxalate deposits and potentially contribute to calcium oxalate renal disease.

Urinary tract infections (UTIs) are a common comorbidity in hospitalized neonates. The current UTI diagnostics have several limitations including invasive collection of urinary samples to ensure sterility, risk of contamination and lack of consensus definitions of UTI based on urine culture. Antimicrobial peptides (AMPs) have been recently utilized as novel biomarkers that can efficiently and accurately diagnose pediatric UTI. However, the concentration of AMPs in neonatal urine is not well-defined. Urine from neonates admitted to a single level IV neonatal intensive care unit was obtained to determine baseline concentration of two AMPs, Ribonuclease 7 (RNase 7) and Beta Defensin-1 (BD-1) and to define the relationship between AMP concentration and gestational age (GA). AMP levels were normalized to urine creatinine. RNase 7 and BD-1 were expressed in neonatal urine (n = 66) regardless of GA and as early as 22 weeks gestation. Urinary concentrations of both AMPs decreased as GA and birthweight increased. The overall median urinary RNase 7/UCr and BD-1/UCr values were 271 ng/mg, and 116 ng/mg, respectively. Median urinary concentrations of RNase 7/UCr for infants born at < 27, 27–32, 33–35 and ≥ 36 weeks were 569, 308, 254, and 124 ng/mg respectively. Similarly, the concentrations of BD-1/UCr at these GA were 166, 115, 108, and 14 ng/mg, respectively. Baseline neonatal urinary concentration of two AMPs (RNase 7 and BD-1) and the variation by GA were identified. This is an essential first step toward the potential utilization of AMPs in improving neonatal UTI diagnostics.

Recent evidence suggests antimicrobial peptides protect the urinary tract from infection. Ribonuclease 7 (RNase 7), a member of the RNase A superfamily, is a potent epithelial-derived protein that maintains human urinary tract sterility. RNase 7 expression is restricted to primates, limiting evaluation of its antimicrobial activity in vivo. Here we identified ribonuclease 6 (RNase 6) as the RNase A superfamily member present in humans and mice that is most conserved at the amino acid level relative to RNase 7. Like RNase 7, recombinant human and murine RNase 6 has potent antimicrobial activity against uropathogens. Quantitative real-time PCR and immunoblot analysis indicate that RNase 6 mRNA and protein are upregulated in the human and murine urinary tract during infection. Immunostaining located RNase 6 to resident and infiltrating monocytes, macrophages, and neutrophils. Uropathogenic E. coli induces RNase 6 peptide expression in human CD14+ monocytes and murine bone marrow–derived macrophages. Thus, RNase 6 is an inducible, myeloid-derived protein with markedly different expression from the epithelial-derived RNase 7 but with equally potent antimicrobial activity. Our studies suggest RNase 6 serves as an evolutionarily conserved antimicrobial peptide that participates in the maintenance of urinary tract sterility.

The mechanisms that maintain sterility in the urinary tract are incompletely understood. Recent studies have implicated the importance of antimicrobial peptides (AMP) in protecting the urinary tract from infection. Here, we characterize the expression and relevance of the AMP human alpha-defensin 5 (HD5) in the human kidney and urinary tract in normal and infected subjects. Using RNA isolated from human kidney, ureter, and bladder tissue, we performed quantitative real-time PCR to show that DEFA5, the gene encoding HD5, is constitutively expressed throughout the urinary tract. With pyelonephritis, DEFA5 expression significantly increased in the kidney. Using immunoblot analysis, HD5 production also increased with pyelonephritis. Immunostaining localized HD5 to the urothelium of the bladder and ureter. In the kidney, HD5 was primarily produced in the distal nephron and collecting tubules. Using immunoblot and ELISA assays, HD5 was not routinely detected in non-infected human urine samples while mean urinary HD5 production increased with E.coli urinary tract infection. DEFA5 is expressed throughout the urinary tract in non-infected subjects. Specifically, HD5 is expressed throughout the urothelium of the lower urinary tract and in the collecting tubules of the kidney. With infection, HD5 expression increases in the kidney and levels become detectable in the urine. To our knowledge, our findings represent the first to quantitate HD5 expression and production in the human kidney. Moreover, this is the first report to detect the presence of HD5 in infected urine samples. Our results suggest that HD5 may have an important role in maintaining urinary tract sterility.

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.

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.