Metabolic acidosis exacerbates pyelonephritis in mice prone to vesicoureteral reflux.
ABSTRACT: Acute pyelonephritis is a common, serious bacterial infection in children. The prevalence of acute pyelonephritis is due at least in part to vesicoureteral reflux (VUR). Although an association between abnormalities in electrolyte and acid-base balance and pyelonephritis is common in young children, the impact of metabolic acidosis (MA) on progression of acute pyelonephritis is not fully understood. In this study, the effect of MA on pyelonephritis was studied in C3H mouse strains prone to VUR. MA induced by ammonium chloride supplementation in food specifically impaired clearance of urinary tract infection with uropathogenic Escherichia. coli (UPEC-UTI) in innate immune competent C3H strains (HeOuJ, HeN), whereas kidney UPEC burden in Tlr-4-deficient HeJ mice was unaffected. Antibody-mediated depletion of myeloid cells (monocytes, neutrophil) markedly increased UPEC burden in the bladder and kidney confirming the pivotal role of neutrophils and tissue-resident macrophages in clearance of UPEC-UTI. MA concurrent with UPEC-UTI markedly increased expression of cytokine (TNF?, IL-1?, IL-6) and chemokine (CXCL 1, 2, and 5) mRNA in isolated kidney CD cells and kidney neutrophil infiltrates were increased four- to fivefold compared to normal, UPEC-infected mice. Thus, MA intensified pyelonephritis and increased the risk of kidney injury by impairing clearance of UPEC-UTI and potentiating renal inflammation characterized by an elevated kidney neutrophil infiltrate.
Project description:Ascending bacterial pyelonephritis, a form of urinary tract infection (UTI) that can result in hospitalization, sepsis, and other complications, occurs in ~250,000 US patients annually; uropathogenic Escherichia coli (UPEC) cause a large majority of these infections. Although UTIs are primarily a disease of women, acute pyelonephritis in males is associated with increased mortality and morbidity, including renal scarring, and end-stage renal disease. Preclinical models of UTI have only recently allowed investigation of sex and sex-hormone effects on pathogenesis. We previously demonstrated that renal scarring after experimental UPEC pyelonephritis is augmented by androgen exposure; testosterone exposure increases both the severity of pyelonephritis and the degree of renal scarring in both male and female mice. Activin A is an important driver of scarring in non-infectious renal injury, as well as a mediator of macrophage polarization. In this work, we investigated how androgen exposure influences immune cell recruitment to the UPEC-infected kidney and how cell-specific activin A production affects post-pyelonephritic scar formation. Compared with vehicle-treated females, androgenized mice exhibited reduced bacterial clearance from the kidney, despite robust myeloid cell recruitment that continued to increase as infection progressed. Infected kidneys from androgenized mice harbored more alternatively activated (M2) macrophages than vehicle-treated mice, reflecting an earlier shift from a pro-inflammatory (M1) phenotype. Androgen exposure also led to a sharp increase in activin A-producing myeloid cells in the infected kidney, as well as decreased levels of follistatin (which normally antagonizes activin action). As a result, infection in androgenized mice featured prolonged polarization of macrophages toward a pro-fibrotic M2a phenotype, accompanied by an increase in M2a-associated cytokines. These data indicate that androgen enhancement of UTI severity and resulting scar formation is related to augmented local activin A production and corresponding promotion of M2a macrophage polarization.
Project description:Urinary tract infections (UTIs) occur predominantly in females but also affect substantial male patient populations; indeed, morbidity in complicated UTI is higher in males. Because of technical obstacles, preclinical modeling of UTI in male mice has been limited. We devised a minimally invasive surgical bladder inoculation technique that yields reproducible upper and lower UTI in both male and female mice, enabling studies of sex differences in these infections. Acute uropathogenic Escherichia coli (UPEC) cystitis in C57BL/6 and C3H/HeN males recapitulated the intracellular bacterial community pathway previously shown in females. However, surgically infected females of these strains exhibited more robust bladder cytokine responses and more efficient UPEC control than males. Compared with females, C3H/HeN males displayed a striking predilection for chronic cystitis, manifesting as persistent bacteriuria, high-titer bladder bacterial burdens, and chronic inflammation. Furthermore, males developed more severe pyelonephritis and 100% penetrant renal abscess (a complication that is rare in female mice). These phenotypes were sharply abrogated after castration but restored with exogenous testosterone, suggesting that male susceptibility to UTI is strongly influenced by androgen exposure. These data substantiate the long-standing presumption that anatomic differences in urogenital anatomy confer protection from UTI in males; however, as clinically observed, male sex associated with more severe UTI once these traditional anatomic barriers were bypassed. This study introduces a highly tractable preclinical model for interrogating sex differences in UTI susceptibility and pathogenesis, and illuminates an interplay between host sex and UTI that is more complex than previously appreciated.
Project description:We present a new preclinical model to study treatment, resolution and sequelae of severe ascending pyelonephritis. Urinary tract infection (UTI), primarily caused by uropathogenic <i>Escherichia coli</i> (UPEC), is a common disease in children. Severe pyelonephritis is the primary cause of acquired renal scarring in childhood, which may eventually lead to hypertension and chronic kidney disease in a small but important fraction of patients. Preclinical modeling of UTI utilizes almost exclusively females, which (in most mouse strains) exhibit inherent resistance to severe ascending kidney infection; consequently, no existing preclinical model has assessed the consequences of recovery from pyelonephritis following antibiotic treatment. We recently published a novel mini-surgical bladder inoculation technique, with which male C3H/HeN mice develop robust ascending pyelonephritis, highly prevalent renal abscesses and evidence of fibrosis. Here, we devised and optimized an antibiotic treatment strategy within this male model to more closely reflect the clinical course of pyelonephritis. A 5-day ceftriaxone regimen initiated at the onset of abscess development achieved resolution of bladder and kidney infection. A minority of treated mice displayed persistent histological abscess at the end of treatment, despite microbiological cure of pyelonephritis; a matching fraction of mice 1?month later exhibited renal scars featuring fibrosis and ongoing inflammatory infiltrates. Successful antibiotic treatment preserved renal function in almost all infected mice, as assessed by biochemical markers 1 and 5?months post-treatment; hydronephrosis was observed as a late effect of treated pyelonephritis. An occasional mouse developed chronic kidney disease, generally reflecting the incidence of this late sequela in humans. In total, this model offers a platform to study the molecular pathogenesis of pyelonephritis, response to antibiotic therapy and emergence of sequelae, including fibrosis and renal scarring. Future studies in this system may inform adjunctive therapies that may reduce the long-term complications of this very common bacterial infection.
Project description:Delta neutrophil index (DNI) is the fraction of circulating immature granulocytes, which reflects severe bacterial infections and septic condition but has not been studied in urinary tract infection (UTI). Here, we evaluated the value of DNI in predicting acute pyelonephritis (APN) or vesicoureteral reflux (VUR) using the data of 288 patients. Conventional inflammatory markers (white blood cell [WBC] count, erythrocyte sedimentation rate [ESR], C-reactive protein [CRP]), and DNI were measured. WBC, CRP, ESR and DNI were higher in APN than in lower UTI (p?<?0.01). Multiple logistic-regression analyses showed that DNI was a predictive factor for areas of lack of uptake on dimercaptosuccinic acid (DMSA) scans (P?<?0.01). The area under the receiver operating characteristic (AUC) was also high for DNI (0.622, 95% CI 0.558-0.687, P?<?0.01) as well as for CRP (0.731, 95% CI 0.673-0.789, P?<?0.01) for the prediction of DMSA defects. DNI demonstrated the highest area under the ROC curve for diagnosis of VUR (0.620, 95% CI 0.542-0.698, P?<?0.01). To the best of our knowledge, this is a first study demonstrating that DNI can be used as a diagnostic marker to distinguish APN from lower UTI and function as a diagnostic marker indicative of VUR compared to other conventional markers.
Project description:Ascending urinary tract infection (UTI) and pyelonephritis caused by uropathogenic Escherichia coli (UPEC) are very common infections that can cause severe kidney damage. Collecting duct cells, the site of hormonally regulated ion transport and water absorption controlled by vasopressin, are the preferential intrarenal site of bacterial adhesion and initiation of inflammatory response. We investigated the effect of the potent V2 receptor (V2R) agonist deamino-8-D-arginine vasopressin (dDAVP) on the activation of the innate immune response using established and primary cultured collecting duct cells and an experimental model of ascending UTI. dDAVP inhibited Toll-like receptor 4-mediated nuclear factor kappaB activation and chemokine secretion in a V2R-specific manner. The dDAVP-mediated suppression involved activation of protein phosphatase 2A and required an intact cystic fibrosis transmembrane conductance regulator Cl- channel. In vivo infusion of dDAVP induced a marked fall in proinflammatory mediators and neutrophil recruitment, and a dramatic rise in the renal bacterial burden in mice inoculated with UPECs. Conversely, administration of the V2R antagonist SR121463B to UPEC-infected mice stimulated both the local innate response and the antibacterial host defense. These findings evidenced a novel hormonal regulation of innate immune cellular activation and demonstrate that dDAVP is a potent modulator of microbial-induced inflammation in the kidney.
Project description:Vesicoureteric reflux (VUR) is a common congenital defect of the urinary tract that is usually discovered after a child develops a urinary tract infection. It is associated with reflux nephropathy, a renal lesion characterized by the presence of chronic tubulointersitial inflammation and fibrosis. Most patients are diagnosed with reflux nephropathy after one or more febrile urinary tract infections, suggesting a potential role for infection in its development. We have recently shown that the C3H mouse has a 100% incidence of VUR. Here, we evaluate the roles of VUR and uropathogenic Escherichia coli infection in the development of reflux nephropathy in the C3H mouse. We find that VUR in combination with sustained kidney infection is crucial to the development of reflux nephropathy, whereas sterile reflux alone fails to induce reflux nephropathy. A single bout of kidney infection without reflux fails to induce reflux nephropathy. The host immune response to infection was examined in two refluxing C3H substrains, HeN and HeJ. HeJ mice, which have a defect in innate immunity and bacterial clearance, demonstrate more significant renal inflammation and reflux nephropathy compared with HeN mice. These studies demonstrate the crucial synergy between VUR, sustained kidney infection and the host immune response in the development of reflux nephropathy in a mouse model of VUR.
Project description:The epidemiology and bacteriology of urinary tract infection (UTI) varies across the human lifespan, but the reasons for these differences are poorly understood. Using established monomicrobial and polymicrobial murine UTI models caused by uropathogenic Escherichia coli (UPEC) and/or Group B Streptococcus (GBS), we demonstrate age and parity as inter-related factors contributing to UTI susceptibility. Young nulliparous animals exhibited 10-100-fold higher bacterial titers compared to older animals. In contrast, multiparity was associated with more severe acute cystitis in older animals compared to age-matched nulliparous controls, particularly in the context of polymicrobial infection where UPEC titers were ?1000-fold higher in the multiparous compared to the nulliparous host. Multiparity was also associated with significantly increased risk of chronic high titer UPEC cystitis and ascending pyelonephritis. Further evidence is provided that the increased UPEC load in multiparous animals required TLR4-signaling. Together, these data strongly suggest that the experience of childbearing fundamentally and permanently changes the urinary tract and its response to pathogens in a manner that increases susceptibility to severe UTI. Moreover, this murine model provides a system for dissecting these and other lifespan-associated risk factors contributing to severe UTI in at-risk groups.
Project description: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 (RegIII?) as the most transcriptionally up-regulated AMP in murine bladder transcriptomes following uropathogenic Escherichia coli (UPEC) infection. We confirmed induction of RegIII? mRNA during cystitis and pyelonephritis by quantitative RT-PCR. Immunoblotting demonstrates increased bladder and urinary RegIII? protein levels following UPEC infection. Immunostaining localizes RegIII? 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 RegIII? 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 RegIII? knockout mice and wild-type mice contain comparable bacterial burden following UPEC and Gram-positive UTI. Our results demonstrate that RegIII? and HIP/PAP expression is induced during human and murine UTI. However, their specific function in the urinary tract remains uncertain.
Project description:Urinary tract infections (UTIs) caused by uropathogenic <i>Escherichia coli</i> (UPEC) induce cystitis, pyelonephritis, and can cause kidney scarring and failure if inflammation is not under control. The detailed effects of cytotoxic necrotizing factor 1 (CNF1), the key UPEC toxin, on the pathogenicity of UPEC remain unclear. CD36 is an important scavenger receptor, responsible for pathogen and apoptotic cell clearance, and plays an essential role in host immune defense and homeostasis. Regulation of CD36 by bacterial toxins has not been reported. In this study, using a pyelonephritis mouse model, CNF1 was observed to contribute to increasing neutrophils and bacterial titers in infected bladder and kidney tissues, resulting in severe inflammation and tissue damage. CD36 expression in macrophages was found to be decreased by CNF1 <i>in vitro</i> and <i>in vivo</i>. We demonstrated that CNF1 attenuated CD36 transcription by decreasing expressions of its upstream transcription factors LXR? and C/EBP? and their recruitment to the CD36 promotor. In addition, Cdc42 was found to be involved in CNF1-mediated downregulation of LXR?. Our study investigated the pathogenesis of <i>cnf1</i>-carrying UPEC, which affected host innate immune defenses and homeostasis through regulation of CD36 in macrophages during acute UTIs.
Project description:Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs), inducing acute pyelonephritis and may result in permanent renal scarring and failure. Alpha-hemolysin (HlyA), a key UPEC toxin, causes serious tissue damage; however, the mechanism through which HlyA induces kidney injury remains unclear. In the present study, granulocyte-macrophage colony-stimulating factor (GM-CSF) secreted by renal epithelial cells was upregulated by HlyA in vitro and in vivo, which induced M1 macrophage accumulation in kidney, and ADAM10 was found involved in HlyA-induced GM-CSF. Macrophage elimination or GM-CSF neutralization protected against acute kidney injury in mice, and increased GM-CSF was detected in urine of patients infected by hlyA-positive UPEC. In addition, HlyA was found to promote UPEC invasion into renal epithelial cells by interacting with Nectin-2 in vitro. However, HlyA did not affect bacterial titers during acute kidney infections, and HlyA-induced invasion did not contribute to GM-CSF upregulation in vitro, which indicate that HlyA-induced GM-CSF is independent of bacteria invasion. The role of GM-CSF in HlyA-mediated kidney injury may lead to novel strategies to treat acute pyelonephritis.