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.

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.

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.

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.

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.

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.

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.

Trans IL-6 signaling does not appear to play a role in renal scarring after urinary tract infection.

INTRODUCTION: While inflammation is an important innate defense mechanism against infection, it can also lead to local tissue damage. The trans signaling pathway of interleukin (IL)-6 is a known mediator of inflammation. We hypothesized that the trans IL-6 signaling pathway is associated with the development of post febrile urinary tract infection (UTI) renal scarring. OBJECTIVE: To compare soluble regulators of trans IL-6 signaling between patients with a history of febrile UTI who do or do not have renal scarring. STUDY DESIGN: After IRB-approval, we collected urine samples in pediatric patients with a history of febrile (≥38 °C) UTI (urine culture >50 K uropathogen) with documented presence or absence of renal scarring on imaging. Samples were collected at a time when patients were not actively infected. Enzyme-linked immunosorbent assays were performed on samples for markers of trans IL-6 signaling: IL-6, soluble (s) IL-6 receptor (R), and soluble (s)gp130, a buffer in trans IL-6 signaling. Values were normalized to urine creatinine. Results were analyzed by t-test or Mann-Whitney U. Spearman rank correlation was used. A p-value of <0.05 was considered significant. RESULTS: A total of 50 urines from patients with a history of febrile UTI were collected: 23 with and 27 without scarring. There was no difference between groups regarding age or gender. There was no significant difference in urine IL-6, sIL-6R, or sgp130 between those with and without scarring (Figure). While IL-6 values significantly correlated with sIL-6R and sgp130 in those without renal scarring, IL-6 did not correlate with sgp130 in those with scarring. Ratios of IL-6 to sgp130 and sIL-6R to sgp130 were not different between groups. DISCUSSION: The inflammatory response generated in response to infection is believed to be largely responsible for the development of renal scarring after UTI. IL-6 is a cytokine known to be induced during UTI with a pro-inflammatory pathway, known as trans signaling. This study investigated for differences in markers of trans IL-6 signaling between patients with a history of febrile UTI with and without renal scarring. There was no significant difference between the absolute values or ratio of these markers between groups. CONCLUSIONS: Markers of trans IL-6 signaling are not different between individuals with a history of febrile UTI with and without renal scarring in the non-acute setting.

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.

Impact of successful pediatric ureteropelvic junction obstruction surgery on urinary HIP/PAP and BD-1 levels.

INTRODUCTION: In the pediatric patient whose ureteropelvic junction obstruction (UPJO) is not always symptomatic, imaging is the most common means of detecting surgical success. There is interest, however, in other means of post-operative monitoring. A panel of antimicrobial peptides (AMPs) has been previously found to be elevated in UPJO, but the impact of surgical correction on these AMPs is unknown. OBJECTIVE: To determine if elevated levels of candidate urinary AMP biomarkers of urinary tract obstruction decrease following UPJO repair. STUDY DESIGN: Pediatric patients undergoing surgical correction of an UPJO were recruited for participation. Bladder urine from uninfected consenting/assenting patients was collected immediately prior to surgery and then at least 6 months afterward. Based on prior studies demonstrating significant elevation of beta defensin 1 (BD-1), hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP), cathelicidin (LL-37), and neutrophil gelatinase-associated lipocalin (NGAL) in patients with UPJO versus control patients, we performed enzyme-linked immunosorbent assays on these four AMPs to compare their expression before and after surgical intervention. If found to significantly decrease, AMP levels were compared to healthy controls. AMP levels were normalized to urine creatinine. Results were analyzed with paired t test or Wilcoxon test using Graphpad software. Correlation was calculated using Pearson or Spearman correlation. A p-value of <0.05 was considered significant. RESULTS: 13 UPJO patients were included in this study; 9 were male (69%). Age at surgery was a median of 4.3 years (average 6.1, range 0.4-18.4 years). Follow-up urine samples were collected a median of 27.4 months after surgery (average 27.4; range 7.8-45.3 months). All 13 patients had clinical improvement and/or signs of improved hydronephrosis on post-operative imaging. HIP/PAP and BD-1 significantly decreased in post-surgical samples compared to pre-surgical samples (p = 0.02 and 0.01, respectively); NGAL and LL-37 did not significantly change. Overall, HIP/PAP decreased in 12 patients (92%) and BD-1 decreased in 11 patients (85%). BD-1 levels after successful repair were not different from healthy controls (p = 0.06). DISCUSSION: Urinary biomarkers of obstruction should detect significant obstructive pathology as well as reflect its resolution. This would enable their use in post-operative monitoring and augment current methods of determining successful surgical outcome through imaging. CONCLUSIONS: The AMPs HIP/PAP and BD-1 are significantly elevated in UPJO but then significantly decrease after pyeloplasty, with BD-1 returning to healthy control levels. As a result, these AMPs could serve as markers of successful surgical intervention.

Differentiating Asymptomatic Bacteriuria From Urinary Tract Infection in the Pediatric Neurogenic Bladder Population: NGAL As a Promising Biomarker.

Objective: To evaluate whether urinary antimicrobial peptides (AMPs) can discriminate between asymptomatic bacteriuria (ASB) and urinary tract infection (UTI) in pediatric patients with neurogenic bladder (NGB). Design/Methods: Bladder urine was collected from pediatric patients (≤18 years old) with NGB without augmentation cystoplasty. Patients were divided into the following groups based on symptomatology and results of urinalysis/urine culture: (a) UTI, (b) ASB, and (c) sterile. Urine AMPs ß defense 1 (BD-1), neutrophil gelatinase-associated lipocalin (NGAL), cathelicidin (LL-37), hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP), and human α defensin 5 (HD-5) were compared between groups by enzyme-linked immunosorbent assays. In addition, urines from pediatric controls without NGB or UTI were also analyzed. Significance was determined using Student's t test for parametric or Mann-Whitney U test for nonparametric data. A p value of <.05 was considered significant. Results: Thirty-six patients with NGB from a spinal dysraphism were evaluated: UTI, n = 6; ASB, n = 18; sterile, n = 12. These groups did not differ significantly by age but did significantly differ by gender (p = .0139). NGAL significantly differed between UTI and ASB groups (median 38.5 ng/mg vs 15.5 ng/mg, respectively; p = .0197) with a sensitivity and specificity of 82.4% and 83.3%, respectively. HIP/PAP, BD-1, HD-5, LL-37, and NGAL levels were all significantly higher in sterile NGB urines compared to 17 non-NGB pediatric controls (p < .0001, p = .0020, p = .0035, p = .0006, and p = .0339, respectively). Conclusion: All five urinary AMPs evaluated were significantly elevated in NGB patients compared to controls. NGAL levels may help differentiate between UTI and ASB in pediatric NGB patients.

Implications of Bacteriuria in Myelomeningocele Patients at Time of Urodynamic Testing.

Objective: To identify those myelomeningocele (MMC) patients at risk for post-urodynamic study (UDS) complications. We hypothesized that patients who manage their bladder with clean intermittent catheterization (CIC) would have a greater risk of post-instrumentation complications due to higher rates of bacteriuria compared to those who freely void (FV). Design/Methods: Urine was collected from patients with MMC without augmentation cystoplasty undergoing routine renal ultrasound or urodynamic study (UDS). Samples were divided into those with bacteriuria (urine culture ≥10,000 colony-forming units) and those without. Post-UDS complications were evaluated and compared between CIC and FV patients. Results: A total of 91 urine samples from 82 total MMC patients were included for evaluation. Significantly more patients on CIC than those who FV had bacteriuria (67% vs 33%, p = .0457). From these urine samples, 54 were obtained at time of UDS of which 45 were from patients on CIC and 9 from FV patients. More patients on CIC had bacteriuria at the time of UDS than those who FV (60% vs 33%, respectively), but this did not reach significance (p = .1416). No patient with bacteriuria on CIC had a complication after UDS while one FV patient with bacteriuria developed post-UDS pyelonephritis. Conclusion: MMC patients with bacteriuria on CIC did not have post-UDS complications. Patients with bacteriuria who FV may be at particular risk for post-instrumentation UTI, providing guidance as to which MMC patients should undergo urine testing prior to UDS in order to prevent post-instrumentation pyelonephritis.

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.

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.

Whole Transcriptome Analysis of Renal Intercalated Cells Predicts Lipopolysaccharide Mediated Inhibition of Retinoid X Receptor alpha Function.

The renal collecting duct consists of intercalated cells (ICs) and principal cells (PCs). We have previously demonstrated that collecting ducts have a role in the innate immune defense of the kidney. Transcriptomics is an important tool used to enhance systems-level understanding of cell biology. However, transcriptomics performed on whole kidneys provides limited insight of collecting duct cell gene expression, because these cells comprise a small fraction of total kidney cells. Recently we generated reporter mouse models to enrich collecting duct specific PC and ICs and reported targeted gene expression of anti-microbial peptide genes. Here we report transcriptomics on enriched ICs and PCs and performed a pilot study sequencing four single ICs. We identified 3,645 genes with increased relative expression in ICs compared to non-ICs. In comparison to non-PCs, 2,088 genes had higher relative expression in PCs. IC associated genes included the innate interleukin 1 receptor, type 1 and the antimicrobial peptide(AMP) adrenomedullin. The top predicted canonical pathway for enriched ICs was lipopolysaccharide/Interleukin 1 mediated inhibition of Retinoid X Receptor alpha function and decreased Retinoid X Receptor expression was confirmed to occur 1-hour post experimental murine UTI in ICs but not in non-ICs.

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.

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.

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.

X-Linked Glomerulopathy Due to COL4A5 Founder Variant.

Alport syndrome is a rare hereditary disorder caused by rare variants in 1 of 3 genes encoding for type IV collagen. Rare variants in COL4A5 on chromosome Xq22 cause X-linked Alport syndrome, which accounts for ∼80% of the cases. Alport syndrome has a variable clinical presentation, including progressive kidney failure, hearing loss, and ocular defects. Exome sequencing performed in 2 affected related males with an undefined X-linked glomerulopathy characterized by global and segmental glomerulosclerosis, mesangial hypercellularity, and vague basement membrane immune complex deposition revealed a COL4A5 sequence variant, a substitution of a thymine by a guanine at nucleotide 665 (c.T665G; rs281874761) of the coding DNA predicted to lead to a cysteine to phenylalanine substitution at amino acid 222, which was not seen in databases cataloguing natural human genetic variation, including dbSNP138, 1000 Genomes Project release version 01-11-2004, Exome Sequencing Project 21-06-2014, or ExAC 01-11-2014. Review of the literature identified 2 additional families with the same COL4A5 variant leading to similar atypical histopathologic features, suggesting a unique pathologic mechanism initiated by this specific rare variant. Homology modeling suggests that the substitution alters the structural and dynamic properties of the type IV collagen trimer. Genetic analysis comparing members of the 3 families indicated a distant relationship with a shared haplotype, implying a founder effect.