Repurposing HDAC inhibitors to enhance ribonuclease 4 and 7 expression and reduce urinary tract infection.

With the emergence of antibiotic-resistant bacteria, innovative approaches are needed for the treatment of urinary tract infections. Boosting antimicrobial peptide expression may provide an alternative to antibiotics. Here, we developed reporter cell lines and performed a high-throughput screen of clinically used drugs to identify compounds that boost ribonuclease 4 and 7 expression (RNase 4 and 7), peptides that have antimicrobial activity against antibiotic-resistant uropathogens. This screen identified histone deacetylase (HDAC) inhibitors as effective RNase 4 and RNase 7 inducers. Validation studies in primary human kidney and bladder cells confirmed pan-HDAC inhibitors as well as the HDAC class I inhibitor, MS-275, induce RNase 4 and RNase 7 to protect human kidney and bladder cells from uropathogenic Escherichia coli. When we administered MS-275 to mice, RNase 4 and 7 expression increased and mice were protected from acute transurethral E. coli challenge. In support of this mechanism, MS-275 treatment increased acetylated histone H3 binding to the RNASE4 and RNASE7 promoters. Overexpression and knockdown of HDAC class I proteins identified HDAC3 as a primary regulator of RNase 4 and 7. These results demonstrate the protective effects of enhancing RNase 4 and RNase 7, opening the door to repurposing medications as antibiotic conserving therapeutics for urinary tract infection.

Keratin 5 basal cells are temporally regulated developmental and tissue repair progenitors in bladder urothelium.

Urothelium forms a distensible yet impermeable barrier, senses and transduces stimuli, and defends the urinary tract from mechanical, chemical, and bacterial injuries. Biochemical and genetic labeling studies support the existence of one or more progenitor populations with the capacity to rapidly regenerate the urothelium following injury, but slow turnover, a low mitotic index, and inconsistent methodologies obscure progenitor identity. The progenitor properties of basal keratin 5 urothelial cells (K5-UCs) have been previously investigated, but those studies focused on embryonic or adult bladder urothelium. Urothelium undergoes desquamation and apoptosis after birth, which requires postnatal proliferation and restoration. Therefore, we mapped the fate of bladder K5-UCs across postnatal development/maturation and following administration of cyclophosphamide to measure homeostatic and reparative progenitor capacities, respectively. In vivo studies demonstrate that basal K5-UCs are age-restricted progenitors in neonates and juveniles, but not in adult mice. Neonatal K5-UCs retain a superior progenitor capacity in vitro, forming larger and more differentiated urothelial organoids than adult K5-UCs. Accordingly, K5-UC transcriptomes are temporally distinct, with enrichment of transcripts associated with cell proliferation and differentiation in neonates. Induction of urothelial proliferation is sufficient to restore adult K5-UC progenitor capacity. Our findings advance the understanding of urothelial progenitors and support a linear model of urothelial formation and regeneration, which may have significant impact on therapeutic development or tissue engineering strategies.NEW & NOTEWORTHY Fate mapping reveals an important linear relationship, whereby bladder basal urothelial cells give rise to intermediate and superficial cells in an age-restricted manner and contribute to tissue repair. Neonatal basal cells reprise their role as superior progenitors in vitro and display distinct transcriptional signatures, which suggest progenitor function is at least partially cell intrinsic. However, the urothelium progenitor niche cannot be overlooked, since FGF7 rescues adult basal cell progenitor function.

Hypertension in children with congenital anomalies of the kidney and urinary tract.

BACKGROUND: Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of childhood chronic kidney disease (CKD). We hypothesized that hypertension varies across CAKUT categories and increases the risk of CKD. METHODS: This was a retrospective cohort study and included cases with a multicystic dysplastic kidney (MCDK, n = 81), unilateral kidney agenesis (UKA, n = 47), kidney hypoplasia (KH, n = 130), and posterior urethral valves (PUV, n = 75). Hypertension was defined as systolic or diastolic blood pressure ≥ 95th percentile for age, sex and height, and CKD as an estimated glomerular filtration rate < 60 ml/min/1.73 m2, both at 2 consecutive clinic visits at least 3 months apart. RESULTS: Sixty-two (19%) out of 333 cases developed hypertension, with significant difference according to CAKUT type. Patients with smaller kidney size (7.7 vs. 8.3, p = 0.045), kidney anomalies in addition to the primary diagnosis (aCAKUT) (53 vs. 38%, p = 0.03), proteinuria (46 vs. 12%, p < 0.001), and CKD (51 vs. 23%, p < 0.001) were more likely to develop hypertension. When adjusted for kidney size, the diagnoses of PUV (OR 10.9, 95%CI 3.0, 40.5), UKA (OR 6.4, 95%CI 1.6, 24.9) and KH (OR 4.2, 95%CI 1.1, 16.1), and aCAKUT (OR 2.1, 95%CI 1.2, 3.9) were independent risk factors for hypertension. Hypertension increased the risk of developing CKD by twofold (HR 1.9, 95%CI 1.19, 2.94). CONCLUSION: Hypertension is common in children with CAKUT and increases the risk of CKD. These findings will aid in the development of a standardized clinical pathway for the care of hypertensive children with CAKUT.

Müllerian anomalies in girls with congenital solitary kidney.

BACKGROUND: The prevalence of Müllerian anomalies (MA) among patients with congenital solitary functioning kidney (SFK) is not well defined. A delay in diagnosis of obstructive MA can increase the risk of poor clinical outcomes. This study describes the prevalence of MA in patients with congenital SFK. METHODS: A retrospective review was performed of patients within the Nationwide Children's Hospital system with ICD9 or ICD10 diagnostic codes for congenital SFK defined as either unilateral renal agenesis (URA) or multicystic dysplastic kidney (MCDK) and confirmed by chart review. Patients with complex urogenital pathology were excluded. Renal anomaly, MA, reason for and type of pelvic evaluation, and age of diagnosis of anomalies were evaluated. RESULTS: Congenital SFK occurred in 431 girls due to URA (209) or MCDK (222). Pelvic evaluation, most commonly by ultrasound for evaluation of abdominal pain or dysmenorrhea, occurred in 115 patients leading to MA diagnosis in 60 instances. Among 221 patients ages 10 years and older, 104 underwent pelvic evaluation and 52 were diagnosed with an MA of which 20 were obstructive. Isolated uterine or combined uterine and vaginal anomalies were the most common MA. MA were five-fold more common in patients with URA compared to MCDK. In 75% of patients, the SFK was diagnosed prior to the MA. CONCLUSIONS: The prevalence of MA in patients with congenital SFK was 24% among those age 10 years or older, and 38% were obstructive. This justifies routine screening pelvic ultrasound in girls with congenital SFK to improve early diagnosis.

A Common Polymorphism in RNASE6 Impacts Its Antimicrobial Activity toward Uropathogenic Escherichia coli.

Human Ribonuclease (RNase) 6 is a monocyte and macrophage-derived protein with potent antimicrobial activity toward uropathogenic bacteria. The RNASE6 gene is heterogeneous in humans due to the presence of single nucleotide polymorphisms (SNPs). RNASE6 rs1045922 is the most common non-synonymous SNP, resulting in a G to A substitution that determines an arginine (R) to glutamine (Q) transversion at position 66 in the protein sequence. By structural analysis we observed that R66Q substitution significantly reduces the positive electrostatic charge at the protein surface. Here, we generated both recombinant RNase 6-R66 and -Q66 protein variants and determined their antimicrobial activity toward uropathogenic Escherichia coli (UPEC), the most common cause of UTI. We found that the R66 variant, encoded by the major SNP rs1045922 allele, exhibited superior bactericidal activity in comparison to the Q66 variant. The higher bactericidal activity of R66 variant correlated with an increase in the protein lipopolysaccharide binding and bacterial agglutination abilities, while retaining the same enzymatic efficiency. These findings encourage further work to evaluate RNASE6 SNP distribution and its impact in UTI susceptibility.

Congenital anomalies of the kidney and urinary tract: defining risk factors of disease progression and determinants of outcomes.

Congenital anomalies of the kidney and urinary tract (CAKUT) result from disruptions in normal kidney and urinary tract development during fetal life and collectively represent the most common cause of kidney failure in children worldwide. The antenatal determinants of CAKUT are diverse and include mutations in genes responsible for normal nephrogenesis, alterations in maternal and fetal environments, and obstruction within the normal developing urinary tract. The resultant clinical phenotypes are complex and depend on the timing of the insult, the penetrance of underlying gene mutations, and the severity and timing of obstruction related to the sequence of normal kidney development. Consequently, there is a broad spectrum of outcomes for children born with CAKUT. In this review, we explore the most common forms of CAKUT and those most likely to develop long-term complications of their associated kidney malformations. We discuss the relevant outcomes for the different forms of CAKUT and what is known about clinical characteristics across the CAKUT spectrum that are risk factors of long-term kidney injury and disease progression.

Predicting outcomes in children with congenital anomalies of the kidney and urinary tract.

BACKGROUND: Congenital anomalies of the kidney and urinary tract (CAKUT) are the most frequent causes of childhood chronic kidney disease (CKD). Using a large CAKUT cohort, we sought to identify the predictors of CKD and to develop a prediction model that informs a risk-stratified clinical pathway. METHODS: This was a retrospective cohort study including cases with multicystic dysplastic kidneys (MCDK), unilateral kidney agenesis (UKA), kidney hypoplasia (KH), and posterior urethral valves (PUV). We identified risk factors for CKD (estimated glomerular filtration rate [eGFR] <60 ml/min/1.73 m2) and tested their performance in an adjusted multivariate binary regression model. Prediction probability scores for CKD were used to separate cases likely to develop complications from those not needing specialist follow-up. RESULTS: We identified 452 eligible cases of CAKUT with 22% developing CKD. Strongest associations with CKD included primary diagnosis (OR 3.5, 95% CI 2.6-4.6), preterm delivery (OR 2.3, 95% CI 1.2-4.4), non-kidney anomalies (OR 1.8, 95% CI 1.1-3), first eGFR<90 (OR 8.9, 95% CI 4.4-18.1), small kidney size (OR 9, 95% CI 4.9-16.6), and additional kidney anomalies (OR 1.6, 95% CI 1.2-2.8). PUV (OR 4.7, 95% CI 1.5-15.3), first eGFR <90 (OR 4.4, 95% CI 2-9.7), and kidney length to body length ratio <7.9 (OR 4.2, 95% CI 1.9-9.2) were independent predictors of CKD. The regression model had a prediction accuracy of 80% and a prediction probability c-statistic of 0.81. CONCLUSION: Using a large combined CAKUT cohort we identified risk factors for CKD. Our prediction model provides the first steps towards a risk-stratified clinical pathway. A higher resolution version of the Graphical abstract is available as Supplementary information.

Urothelial progenitors in development and repair.

Urothelium is a specialized multilayer epithelium that lines the urinary tract from the proximal urethra to the kidney. In addition to proliferation and differentiation during development, urothelial injury postnatally triggers a robust regenerative capacity to restore the protective barrier between the urine and tissue. Mounting evidence supports the existence of dedicated progenitor cell populations that give rise to urothelium during development and in response to injury. Understanding the cellular and molecular basis for urothelial patterning and repair will inform tissue regeneration therapies designed to ameliorate a number of structural and functional defects of the urinary tract. Here, we review the current understanding of urothelial progenitors and the signaling pathways that govern urothelial development and repair. While most published studies have focused on bladder urothelium, we also discuss literature on upper tract urothelial progenitors. Furthermore, we discuss evidence supporting existence of context-specific progenitors. This knowledge is fundamental to the development of strategies to regenerate or engineer damaged or diseased urothelium.

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.

Prediction of kidney failure in children with chronic kidney disease and obstructive uropathy.

BACKGROUND: Obstructive uropathy (OU) is a leading cause of pediatric kidney injury. Accurate prediction of kidney disease progression may improve clinical outcomes. We aimed to examine discrimination and accuracy of a validated kidney failure risk equation (KFRE), previously developed in adults, in children with OU. METHODS: We identified 118 children with OU and an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2 in the Chronic Kidney Disease in Children study, a national, longitudinal, observational cohort. Each patient's 5-year risk of kidney failure was estimated using baseline data and published parameters for the 4- and 8-variable KFREs. Discriminative ability of the KFRE was estimated using the C statistic for time-to-event analysis. Sensitivity and specificity were evaluated across varying risk thresholds. RESULTS: Among the 118 children, 100 (85%) were boys, with median baseline age of 10 years (interquartile range, 6-14). Median eGFR was 42 mL/min/1.73m 2 (32-53), with a median follow-up duration of 4.5 years (2.7-7.2); 23 patients (19.5%) developed kidney failure within 5 years. The 4-variable KFRE discriminated kidney failure risk with a C statistic of 0.75 (95% CI, 0.68-0.82). A 4-variable risk threshold of ≥ 30% yielded 82.6% sensitivity and 75.0% specificity. Results were similar using the 8-variable KFRE. CONCLUSIONS: In children with OU, the KFRE discriminated the 5-year risk of kidney failure at C statistic values lower than previously published in adults but comparable with suboptimal values reported in the overall CKiD population. The 8-variable equation did not improve model discrimination or accuracy, suggesting the need for continued research into additional, disease-specific markers.

Innate immunity and urinary tract infection.

Urinary tract infections are a severe public health problem. The emergence and spread of antimicrobial resistance among uropathogens threaten to further compromise the quality of life and health of people who develop acute and recurrent upper and lower urinary tract infections. The host defense mechanisms that prevent invasive bacterial infection are not entirely delineated. However, recent evidence suggests that versatile innate immune defenses play a key role in shielding the urinary tract from invading uropathogens. Over the last decade, considerable advances have been made in defining the innate mechanisms that maintain immune homeostasis in the kidney and urinary tract. When these innate defenses are compromised or dysregulated, pathogen susceptibility increases. The objective of this review is to provide an overview of how basic science discoveries are elucidating essential innate host defenses in the kidney and urinary tract. In doing so, we highlight how these findings may ultimately translate into the clinic as new biomarkers or therapies for urinary tract infection.

Longitudinal kidney injury biomarker trajectories in children with obstructive uropathy.

BACKGROUND: Congenital obstructive uropathy (OU) is a leading cause of pediatric kidney failure, representing a unique mechanism of injury, in part from renal tubular stretch and ischemia. Tubular injury biomarkers have potential to improve OU-specific risk stratification. METHODS: Patients with OU were identified in the Chronic Kidney Disease in Children (CKiD) study. "Cases" were defined as individuals receiving any kidney replacement therapy (KRT), while "controls" were age- and time-on-study matched and KRT free at last study visit. Urine and plasma neutrophil gelatinase-associated lipocalin (NGAL), interleukin 18 (IL-18), and liver-type fatty acid-binding protein (L-FABP) levels were measured at enrollment and annually and compared between cases and controls. Urine values were normalized to urine creatinine. RESULTS: In total, 22 cases and 22 controls were identified, with median (interquartile range) ages of 10.5 (9.0-13.0) and 15.9 (13.9-16.9) years at baseline and outcome, respectively. At enrollment there were no differences noted between cases and controls for any urine (u) or plasma (p) biomarker measured. However, the mean pNGAL and uL-FABP/creatinine increased throughout the study period in cases (15.38 ng/ml per year and 0.20 ng/ml per mg/dl per year, respectively, p = 0.01 for both) but remained stable in controls. This remained constant after controlling for baseline glomerular filtration rate (GFR). CONCLUSIONS: In children with OU, pNGAL and uL-FABP levels increased over the 5 years preceding KRT; independent of baseline GFR. Future studies are necessary to identify optimal cutoff values and to determine if these markers outperform current clinical predictors.

Ribonuclease 7 Shields the Kidney and Bladder from Invasive Uropathogenic Escherichia coli Infection.

BACKGROUND: Evidence suggests that antimicrobial peptides, components of the innate immune response, protect the kidneys and bladder from bacterial challenge. We previously identified ribonuclease 7 (RNase 7) as a human antimicrobial peptide that has bactericidal activity against uropathogenic Escherichia coli (UPEC). Functional studies assessing RNase 7's contributions to urinary tract defense are limited. METHODS: To investigate RNase 7's role in preventing urinary tract infection (UTI), we quantified urinary RNase 7 concentrations in 29 girls and adolescents with a UTI history and 29 healthy female human controls. To assess RNase 7's antimicrobial activity in vitro in human urothelial cells, we used siRNA to silence urothelial RNase 7 production and retroviral constructs to stably overexpress RNase 7; we then evaluated UPEC's ability to bind and invade these cells. For RNase 7 in vivo studies, we developed humanized RNase 7 transgenic mice, subjected them to experimental UTI, and enumerated UPEC burden in the urine, bladder, and kidneys. RESULTS: Compared with controls, study participants with a UTI history had 1.5-fold lower urinary RNase 7 concentrations. When RNase 7 was silenced in vitro, the percentage of UPEC binding or invading human urothelial cells increased; when cells overexpressed RNase 7, UPEC attachment and invasion decreased. In the transgenic mice, we detected RNase 7 expression in the kidney's intercalated cells and bladder urothelium. RNase 7 humanized mice exhibited marked protection from UPEC. CONCLUSIONS: These findings provide evidence that RNase 7 has a role in kidney and bladder host defense against UPEC and establish a foundation for investigating RNase 7 as a UTI prognostic marker or nonantibiotic-based therapy.

Krt5+ urothelial cells are developmental and tissue repair progenitors in the kidney.

Congenital urinary tract obstruction (UTO) is the leading cause of chronic kidney disease in children; however, current management strategies do not safeguard against progression to end-stage renal disease, highlighting the need for interventions to limit or reverse obstructive nephropathy. Experimental UTO triggers renal urothelial remodeling that culminates in the redistribution of basal keratin 5-positive (Krt5+) renal urothelial cells (RUCs) and the generation of uroplakin-positive (Upk)+ RUCs that synthesize a protective apical urothelial plaque. The cellular source of Upk+ RUCs is currently unknown, limiting the development of strategies to promote renal urothelial remodeling as a therapeutic approach. In the present study, we traced the origins of adult Upk+ RUCs during normal development and in response to UTO. Fate mapping analysis demonstrated that adult Upk+ RUCs derive from embryonic and neonatal Krt5+ RUCs, whereas Krt5+ RUCs lose this progenitor capacity and become lineage restricted by postnatal day 14. However, in response to UTO, postnatal day 14-labeled adult Krt5+ RUCs break their lineage restriction and robustly differentiate into Upk+ RUCs. Thus, Krt5+ RUCs drive renal urothelial formation during normal ontogeny and after UTO by differentiating into Upk+ RUCs in a temporally restricted manner.

Interleukin-1beta selectively expands and sustains interleukin-22+ immature human natural killer cells in secondary lymphoid tissue.

Among human natural killer (NK) cell intermediates in secondary lymphoid tissue (SLT), stage 3 CD34(-)CD117(+)CD161(+)CD94(-) immature NK (iNK) cells uniquely express aryl hydrocarbon receptor (AHR) and interleukin-22 (IL-22), supporting a role in mucosal immunity. The mechanisms controlling proliferation and differentiation of these cells are unknown. Here we demonstrate that the IL-1 receptor IL-1R1 was selectively expressed by a subpopulation of iNK cells that localized proximal to IL-1beta-producing conventional dendritic cells (cDCs) within SLT. IL-1R1(hi) iNK cells required continuous exposure to IL-1beta to retain AHR and IL-22 expression, and they proliferate in direct response to cDC-derived IL-15 and IL-1beta. In the absence of IL-1beta, a substantially greater fraction of IL-1R1(hi) iNK cells differentiated to stage 4 NK cells and acquired the ability to kill and secrete IFN-gamma. Thus, cDC-derived IL-1beta preserves and expands IL-1R1(hi)IL-22(+)AHR(+) iNK cells, potentially influencing human mucosal innate immunity during infection.

Common clinical markers predict end-stage renal disease in children with obstructive uropathy.

BACKGROUND: Obstructive uropathy (OU) is a common cause of end-stage renal disease (ESRD) in children. Children who escape the newborn period with mild-to-moderate chronic kidney disease (CKD) continue to be at increased risk. The predictive ability of clinically available markers throughout childhood is poorly defined. METHODS: Patients with OU were identified in the Chronic Kidney Disease in Children Study. The primary outcome of interest was renal replacement therapy (RRT) (cases). Controls were age matched and defined as patients within the OU cohort who did not require RRT during study follow-up. RESULTS: In total, 27 cases and 41 age-matched controls were identified. Median age at baseline and age at outcome measurement were 10 vs. 16 years, respectively. First available glomerular filtration rate (GFR) (36.9 vs. 53.5 mL/min per 1.73 m2), urine protein/creatinine (Cr) (0.40 vs. 0.22 mg/mg) and microalbumin/Cr (0.58 vs. 0.03 mg/mg), and serum CO2 (20 vs. 22 mmol/L) and hemoglobin (12.4 vs. 13.2 g/dL) differed significantly between cases and controls, respectively. GFR declined 3.07 mL/min per 1.73 m2/year faster in cases compared to that in controls (p < 0.0001). Urine protein/Cr and microalbumin/Cr increased by 0.16 and 0.11 per year more in cases compared to those in controls, respectively (p ≤ 0.001 for both). Serum phosphate increased by 0.11 mg/dL and serum albumin and hemoglobin decreased by 0.04 (g/dL) and 0.14 (g/dL) per year more for cases compared to those for controls, respectively (p < 0.05 for all). CONCLUSIONS: Age-specific baseline and longitudinal measures of readily available clinical measures predict progression to ESRD in children with mild-to-moderate CKD from OU.

The uroplakin plaque promotes renal structural integrity during congenital and acquired urinary tract obstruction.

Urinary tract obstruction represents a common cause of kidney injury across the human life span, resulting in chronic kidney disease and end-stage renal disease. Yet, the extent of obstructive renal damage can be heterogeneous between individuals, implying the existence of unknown mechanisms that protect against or accelerate kidney injury. In this study, we investigated the role of urothelial remodeling in renal adaptation during congenital and acquired obstruction. In the Megabladder ( Mgb-/-) model of congenital obstruction and unilateral ureteral ligation model of acute obstruction, progressive hydronephrosis is strongly associated with dynamic reorganization of the renal urothelium, which elaborates a continuous uroplakin (Upk) plaque. This led us to postulate that the Upk plaque prevents parenchymal injury during urinary tract obstruction. To test this hypothesis, we interbred Mgb-/- and Upk1b-/- mice, which lack the critical Upk1b subunit for Upk plaque formation. Upk1b-/-; Mgb-/- mice experienced an accelerated onset of bilateral hydronephrosis with severe (>67%) parenchymal loss, leading to renal failure and mortality in adolescence. To investigate the function of the renal Upk plaque during acute obstruction, we destabilized the Upk plaque by Upk1b deletion or genetically depleted Upk+ cells following unilateral ureteral obstruction. Both of these strategies accelerated renal parenchymal loss following ureteral ligation, attesting to a conserved, stabilizing role for Upk plaque deposition in the acutely obstructed kidney. In aggregate, these complementary experiments provide the first evidence that the Upk plaque confers an essential, protective adaptation to preserve renal parenchymal integrity during congenital and acquired urinary tract obstruction.

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.

Novel role for androgen signaling in pyelonephritis.

Sex differences in urinary tract infection (UTI) susceptibility and severity are known, but have historically focused on anatomic differences between males and females. Until recently, experimental UTI has been limited to female animals due to ease of transurethral bladder catheterization. Olson and colleagues have developed a model of experimental UTI independent of sex that relies on direct bladder inoculation and thus permits investigation of sex differences in UTI susceptibility. They now build upon their prior work in this model by implicating androgens as drivers of tubular invasion by uropathogenic Escherichia coli, which form luminal bacterial communities preceding renal abscess formation.

Interleukin-6/Stat3 signaling has an essential role in the host antimicrobial response to urinary tract infection.

The signaling networks regulating antimicrobial activity during urinary tract infection (UTI) are incompletely understood. Interleukin-6 (IL-6) levels increase with UTI severity, but the specific contributions of IL-6 to host immunity against bacterial uropathogens are unknown. To clarify this we tested whether IL-6 activates the Stat3 transcription factor, to drive a program of antimicrobial peptide gene expression in infected urothelium during UTI. Transurethral inoculation of uropathogenic Escherichia coli led to IL-6 secretion, urothelial Stat3 phosphorylation, and activation of antimicrobial peptide transcription, in a Toll-like receptor 4-dependent manner in a murine model of cystitis. Recombinant IL-6 elicited Stat3 phosphorylation in primary urothelial cells in vitro, and systemic IL-6 administration promoted urothelial Stat3 phosphorylation and antimicrobial peptide expression in vivo. IL-6 deficiency led to decreased urothelial Stat3 phosphorylation and antimicrobial peptide mRNA expression following UTI, a finding mirrored by conditional Stat3 deletion. Deficiency in IL-6 or Stat3 was associated with increased formation of intracellular bacterial communities, and exogenous IL-6 reversed this phenotype in IL-6 knockout mice. Moreover, chronic IL-6 depletion led to increased renal bacterial burden and severe pyelonephritis in C3H/HeOuJ mice. Thus, IL-6/Stat3 signaling drives a transcriptional program of antimicrobial gene expression in infected urothelium, with key roles in limiting epithelial invasion and ascending infection.