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Kidney intercalated cells are involved in acid-base homeostasis via vacuolar ATPase expression. Here we report six human intercalated cell subtypes, including hybrid principal-intercalated cells identified from single cell transcriptomics. Phagosome maturation is a biological process that increases in biological pathway analysis rank following exposure to uropathogenic Escherichia coli in two of the intercalated cell subtypes. Real time confocal microscopy visualization of murine renal tubules perfused with green fluorescent protein expressing Escherichia coli or pHrodo Green E. coli BioParticles demonstrates that intercalated cells actively phagocytose bacteria then acidify phagolysosomes. Additionally, intercalated cells have increased vacuolar ATPase expression following in vivo experimental UTI. Taken together, intercalated cells exhibit a transcriptional response conducive to the kidney’s defense, engulf bacteria and acidify the internalized bacteria. Intercalated cells represent an epithelial cell with characteristics of professional phagocytes like macrophages., Kidney intercalated cells are involved in acid-base homeostasis in the kidneys. Here, the authors use single cell transcriptomics and find that interalated cells exhibit a transcriptional response conducive to defense and can engulf and acidify internalized bacteria, similarly to professional phagocytes.

Urinary tract infections (UTI), associated with vesicoureteral reflux (VUR), can lead to chronic kidney disease. Genetic alterations in the innate immune defenses contribute to UTI risk. We investigated a novel gene, Dachsous Cadherin-Related 1 ( DCHS1), in children with UTI. We determined absolute DNA copy number (CN) of DCHS1 in children with UTI. In this case-control study, we utilized multiple complementary methods to determine the genomic CN of DCHS1. Children with ( n = 370) and without ( n = 71) VUR from two well-phenotyped clinical trials of UTI were copy-typed and compared to 491 healthy controls with no known history of VUR or UTI. Less than 1% of controls had a single copy of DCHS1, while 31% of children with UTI and no VUR and 7% of children with UTI and VUR had a single copy of the DCHS1 gene. Using immunostaining, we localized expression postnatally to the bladder and renal epithelia. Mice were also challenged with two uropathogenic Escherichia coli strains, and Dchs1 mRNA was quantified. This study represents the first report of DCHS1 in association with pediatric UTI. We hypothesize that its role in innate immunity is critical to lower urinary tract defense. Further investigation is required to determine the role of DCHS1 in innate immunity.

Background: The 2011 American Academy of Pediatrics clinical practice guideline recommends when to obtain renal and bladder ultrasound (RBUS) and voiding cystourethrography (VCUG) following febrile urinary tract infection (UTI) for children age 2–24 months. However, there is not consensus about when to obtain imaging in neonates. The objective of this study is to evaluate UTI diagnostic criteria along with RBUS and VCUG in neonates admitted to the NICU in the first 3 months of life. Methods: A retrospective electronic medical record review was performed of neonates admitted to Nationwide Children's Hospital system NICUs between January 2010 and December 2014 with UTI as a primary or secondary diagnosis. Urine culture results were evaluated versus established UTI criteria and renal US and VCUG results were compared. Results: Of 81 patients with a straight catheterized urine culture obtained, 28 patients met laboratory criteria for diagnosis of UTI and all but 4 had a RBUS. Urine cultures had an equal distribution of Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, and Coagulase negative staphylococcus. RBUS showed dilation of the collecting system in 37.5% of patients with UTI compared to 41.3% without UTI. VCUG showed vesicourteral reflux (VUR) on 41.7% of those with UTI compared to 34.8% without UTI. For patients with UTI, the sensitivity of RBUS for VUR on VCUG was 60% with CI [0.17, 0.93] and specificity was 43% with CI [0.12, 0.80]. In patients without UTI, sensitivity of RBUS for VUR on VCUG was 63% with CI [0.26, 0.90] and specificity was 71% with CI [0.42, 0.90]. Conclusions: Fewer than half of neonates that were diagnosed clinically with UTI met laboratory criteria for a UTI. Positive urine cultures grew a wide variety of organisms. The sensitivity of renal ultrasound for VUR is only about 60%. Key Words: cystitis, cystourethrogram, ultrasound, urinalysis

Historically, the role of bacteria in urinary stone disease (USD) has been limited to urease-producing bacteria associated with struvite stone formation. However, growing evidence has revealed bacteria associated with stones of non-struvite composition. These bacteria may be derived from either urine or from the stones themselves. Using 16S rRNA gene sequencing and an enhanced culture technique (EQUC), we identified the urine and stone microbiota of USD patients and then determined if bacteria were statistically enriched in the stones relative to the urine. From 52 patients, bladder urine and urinary stones were collected intraoperatively during ureteroscopy. Stone homogenate and urine specimens were subjected to 16S rRNA gene sequencing and EQUC. Standard Chi-squared tests were applied to determine if the relative abundance of any bacterial taxon was significantly enriched in urinary stones compared to urine. Stones were primarily calcium-based. 29/52 (55.8%) stones had bacteria detected by 16S rRNA gene sequencing. Of these, dominant bacterial taxa were enriched from 12 stones. Bacterial taxa isolated by EQUC include members of the genera Staphylococcus, Enterobacter, Escherichia, Corynebacterium, and Lactobacillus. Dominant bacterial genera were enriched compared to paired bladder urine. Differences between the stone and urine microbiota may indicate that certain bacteria contribute to USD pathophysiology. Further investigation is warranted.

Objectives: To describe variations in coronavirus disease 2019 (COVID-19) diagnosis by zip code race and ethnicity in Indiana.Methods: Cross-sectional evaluation of subjects with SARS-CoV-2 at Indiana University Health. We performed two separate analyses, first evaluating likelihood of COVID-19 diagnosis by race (Caucasian, African American, Asian, or other) and ethnicity (Hispanic vs. non-Hispanic) in the cohort encompassing the entire state of Indiana. Subsequently, patient data was geolocated with zip codes in Marion County and the immediate surrounding counties, and descriptive statistical analyses were used to calculate the number of COVID-19 cases per 10,000 persons for each of these zip codes.Results: Indiana had a total of 3,892 positive COVID-19 cases from January 1 to April 30, 2020. The odds of testing positive for COVID-19 were four-fold higher in African Americans than non-African Americans (OR 4.58, 95% CI 4.25–4.94, P < 0.0001). Increased COVID-19 cases per 10,000 persons were seen in zip codes with higher percentage of African American (median infection rate of 17.4 per 10,000 population in zip codes above median % African American compared to 6.7 per 10,000 population in zip codes below median % African American, with an overall median infection rate 9.9 per 10,000 population, P < 0.0001) or Hispanic residents (median infection rate of 15.9 per 10,000 population in zip codes above median % Hispanic compared to 7.0 per 10,000 population in zip codes below median % Hispanic, overall median infection rate 9.6 per 10,000 population, P < 0.0001).Conclusions: Individuals from zip codes with higher percentages of African American, Hispanic, foreign-born, and/or residents living in poverty are disproportionately affected by COVID-19. Urgent work is needed to understand and address the disproportionate burden of COVID-19 in minority communities and when economic disparities are present.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated disease COVID-19 (coronavirus disease 2019) has presented graduate medical education (GME) training programs with a unique set of challenges. One of the most pressing is how should hospital systems that rely on graduate medical residents provide appropriate care for patients while protecting trainees. This question is of particular concern as healthcare workers are at high risk of SARS-CoV-2 exposure.Objective: This cross-sectional study sought to assess the impact of hospital COVID-19 patient placement on pediatric graduate medical residents by comparing rates of SARS-CoV-2 seroconversion rates of residents who worked on designated COVID-19 teams and those who did not.Methods: Forty-four pediatric and medicine–pediatric residents at Riley Children's Hospital (Indianapolis, IN) were tested for SARS-CoV-2 immunoglobulin M (IgM) and IgG seroconversion in May 2020 using enzyme-linked immunosorbent assays (Abnova catalog no. KA5826), 2 months after the first known COVID-19 case in Indiana. These residents were divided into two groups: those residents who worked on designated COVID-19 teams, and those who did not. Groups were compared using χ2 or Fisher exact test for categorical variables, and continuous variables were compared using Student t testing.Results: Forty-four of 104 eligible residents participated in this study. Despite high rates of seroconversion, there was no difference in the risk of SARS-CoV-2 seroconversion between residents who worked on designated COVID-19 teams (26% or 8/31) and those who did not (31% or 4/13). Eleven of 44 residents (25%) tested positive for SARS-CoV-2 IgG, whereas only 5/44 (11.4%) tested positive for SARS-CoV-2 IgM, without a detectable difference between exposure groups.Conclusion: We did not observe a difference in SARS-CoV-2 seroconversion between different exposure groups. These data are consistent with growing evidence supporting the efficacy of personal protective equipment. Further population-based research on the role of children in transmitting the SARS-CoV-2 virus is needed to allow for a more evidence-based approach toward managing the COVID-19 pandemic.

Background:Acute kidney injury (AKI) due to urinary stone disease (USD) is rare in adults; AKI rates in children with USD may be higher, and emerging data links stones to chronic kidney disease (CKD) development in adults.Methods:This study is a retrospective analysis of USD patients at a single pediatric hospital system's emergency department (ED). Patients were initially identified by USD ICD codes; USD was then confirmed by imaging or physician documentation; patients had to have baseline creatinine (Cr) and Cr in the ED for comparison to be included. AKI was defined by Kidney Disease: Improving Global Outcomes (KDIGO), Acute Kidney Injury Network (AKIN), and Pediatric Risk, Injury, Failure, Loss, End Stage (pRIFLE).Results:Of the 589 total visits, 264/589 (45%) had data to evaluate for AKI, 23% were AKI(+) and 77% were AKI(–). pRIFLE was most common (82%) and 18% were only positive by AKIN/KDIGO. AKI(+) were more likely to be younger (16.7 vs. 17.4 years,p= 0.046) and more likely to present with vomiting {odds ratio [OR] [95% confidence interval (CI)]: 2.4 [1.4–4.3],p= 0.002}; also, the proportion of AKI(+) was significantly higher in p= 0.032, OR (95% CI): 2.0 (1.1–3.9)]. Urinary tract infection (UTI) and obstruction rates were similar between groups. AKI(+) patients had a significant OR p= 0.001).Conclusion:We have demonstrated a novel association between USD-induced renal colic and AKI in a group of young adults and children. AKI(+) patients were younger and were more likely to present with vomiting. AKI(+) patients did not have higher rates of obstruction or UTI, and 51% of AKI(+) received NSAIDs.

BACKGROUND: The occurrence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the associated coronavirus disease 2019 (COVID-19) have profoundly affected adult kidney disease patients. In contrast, pediatric solid organ transplant recipients, including pediatric kidney transplant (KT) recipients, do not seem to be at particularly higher risk for SARS-CoV-2 infection or for severe COVID-19 disease. This patient population might be protected by certain mechanisms, such as the immunosuppressive medications with their anti-inflammatory properties or simply being well-versed in self-protection techniques. Assessing SARS-CoV-2 antibody serologies could potentially help understand why this patient population is apparently spared from severe SARS-CoV-2 clinical courses. OBJECTIVE: To examine SARS-CoV-2 serologic status in a cohort of pediatric KT recipients. METHODS: SARS-CoV-2 anti-spike IgG and IgM antibodies were measured by three different methods in pediatric KT recipients coming for routine clinic visits immediately post-confinement in May-June of 2020. The patients were considered seroconverted if SARS-CoV-2 antibodies were positive by 2/3 methods and weak positive/indeterminate if positive by 1/3. RESULTS: Thirty-one patients were evaluated (about 1/3 of our institution's pediatric KT population). One patient seroconverted, while three were considered weak positive/indeterminate. None were symptomatic and none had nasopharyngeal PCR confirmed SARS-CoV-2 disease. CONCLUSIONS: Seroconversion to SARS-CoV-2 was rare in this population and likely reflects the social distancing practiced by these patients. The results will serve as a foundation for a future longitudinal study to evaluate the long-term emergence and persistence of antibodies in this population and may inform studies of response to a future vaccine.

Dyspnea is the second leading cause of US emergency department (ED) visits and an independent predictor of morbidity and mortality1 in older adult patients aged ≥65 years. Unfortunately, the diagnosis of the cause of dyspnea presents diagnostic challenges to emergency physicians2-4 that disproportionately affects older adults.5 One in 5 dyspneic older adults experience missed diagnosis in the ED2 and 21% are treated for ≥1 pneumonia, acute exacerbation of chronic obstructive pulmonary disease [COPD], and acute exacerbation of heart failure [HF].5 Importantly, some may have multiple causes of their dyspnea but accurate diagnosis remains critical.

Coronavirus disease 2019 (COVID‐19) is a highly infectious disease caused by the severe acute respiratory syndrome coronavirus 2 virus (SARS‐CoV‐2). While children appear to experience less severe disease than adults, those with underlying conditions such as kidney disease may be more susceptible to infection. Limited data are present for children with kidney disease, and there are limited prior reports of pediatric hemodialysis patients with COVID‐19. This report describes the mild clinical disease course of COVID‐19 in two pediatric patients with chronic kidney disease, one on hemodialysis and both on chronic immunosuppression. We review treatment in these patients, as well as our measures to reduce transmission among our hemodialysis patients and staff.

Carbonic anhydrase II knockout ( Car2−/−) mice have depleted numbers of renal intercalated cells, which are increasingly recognized to be innate immune effectors. We compared pyelonephritis susceptibility following reciprocal renal transplantations between Car2−/−and wild-type mice. We examined the effect of pharmacological CA suppression using acetazolamide in an experimental murine model of urinary tract infection. Car2−/−versus wild-type mice were compared for differences in renal innate immunity. In our transplant scheme, mice lacking CA-II in the kidney had increased pyelonephritis risk. Mice treated with acetazolamide had lower kidney bacterial burdens at 6 h postinfection, which appeared to be due to tubular flow from diuresis because comparable results were obtained when furosemide was substituted for acetazolamide. Isolated Car2−/−kidney cells enriched for intercalated cells demonstrated altered intercalated cell innate immune gene expression, notably increased calgizzarin and insulin receptor expression. Intercalated cell number and function along with renal tubular flow are determinants of pyelonephritis risk.

People with diabetes mellitus have increased infection risk. With diabetes, urinary tract infection (UTI) is more common and has worse outcomes. Here, we investigate how diabetes and insulin resistance impact the kidney’s innate defenses and urine sterility. We report that type 2 diabetic mice have increased UTI risk. Moreover, insulin-resistant prediabetic mice have increased UTI susceptibility, independent of hyperglycemia or glucosuria. To identify how insulin resistance affects renal antimicrobial defenses, we genetically deleted the insulin receptor in the kidney’s collecting tubules and intercalated cells. Intercalated cells, located within collecting tubules, contribute to epithelial defenses by acidifying the urine and secreting antimicrobial peptides (AMPs) into the urinary stream. Collecting duct and intercalated cell–specific insulin receptor deletion did not impact urine acidification, suppressed downstream insulin-mediated targets and AMP expression, and increased UTI susceptibility. Specifically, insulin receptor–mediated signaling regulates AMPs, including lipocalin 2 and ribonuclease 4, via phosphatidylinositol-3-kinase signaling. These data suggest that insulin signaling plays a critical role in renal antibacterial defenses.

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.

More than 10% of the global human population is now afflicted with kidney stones, which are commonly associated with other significant health problems including diabetes, hypertension and obesity. Nearly 70% of these stones are primarily composed of calcium oxalate, a mineral previously assumed to be effectively insoluble within the kidney. This has limited currently available treatment options to painful passage and/or invasive surgical procedures. We analyze kidney stone thin sections with a combination of optical techniques, which include bright field, polarization, confocal and super-resolution nanometer-scale auto-fluorescence microscopy. Here we demonstrate using interdisciplinary geology and biology (geobiology) approaches that calcium oxalate stones undergo multiple events of dissolution as they crystallize and grow within the kidney. These observations open a fundamentally new paradigm for clinical approaches that include in vivo stone dissolution and identify high-frequency layering of organic matter and minerals as a template for biomineralization in natural and engineered settings.

Urinary stone disease (USD) is affecting a greater number of children and low bone mineral density (BMD) and increased skeletal fractures have been demonstrated in stone patients; however, the mechanism(s) driving bone disease remain unclear. This pilot study was undertaken to assess an adolescent kidney stone cohort’s BMD and evaluate for an inverse correlation between BMD and urine concentration of lithogenic minerals and/or inflammatory levels. Prospective case–control study was carried out at a large pediatric center. 15 participants with USD (12–18 years of age, 8 female) were matched by age, sex, and body mass index to 15 controls. Lumbar and total body BMD z-score did not differ between groups. When stone formers were separated by sex, there was a significant difference between male stone formers vs. controls total body BMD z-score (Fig. 1). BMD z-score did not significantly correlate with urine calcium, oxalate, citrate or magnesium. Higher urine IL-13 did significantly correlate with higher total body BMD z-score (r = 0.677, p = 0.018). Total body BMD z-score did significantly correlate with body mass index (BMI) as expected for the control group (r = 0.6321, p = 0.0133). However, this relationship was not present in the USD group (r = − 0.1629, p = 0.5619). This is a small but hypothesis-generating study which demonstrates novel evidence of male-specific low BMD in adolescent stone formers. Furthermore, we demonstrated a positive association between urine IL-13 and total body BMD z-score USD patients as well as a lack of a positive BMD and BMI correlations in stone formers.

The incidence of nephrolithiasis continues to rise. Previously, we showed that a monogenic cause could be detected in 11.4% of individuals with adult-onset nephrolithiasis or nephrocalcinosis and in 16.7-20.8% of individuals with onset before 18 years of age, using gene panel sequencing of 30 genes known to cause nephrolithiasis/nephrocalcinosis. To overcome the limitations of panel sequencing, we utilized whole exome sequencing in 51 families, who presented before age 25 years with at least one renal stone or with a renal ultrasound finding of nephrocalcinosis to identify the underlying molecular genetic cause of disease. In 15 of 51 families, we detected a monogenic causative mutation by whole exome sequencing. A mutation in seven recessive genes ( AGXT, ATP6V1B1, CLDN16, CLDN19, GRHPR, SLC3A1, SLC12A1 ), in one dominant gene ( SLC9A3R1 ), and in one gene ( SLC34A1 ) with both recessive and dominant inheritance was detected. Seven of the 19 different mutations were not previously described as disease-causing. In one family, a causative mutation in one of 117 genes that may represent phenocopies of nephrolithiasis-causing genes was detected. In nine of 15 families, the genetic diagnosis may have specific implications for stone management and prevention. Several factors that correlated with the higher detection rate in our cohort were younger age at onset of nephrolithiasis/nephrocalcinosis, presence of multiple affected members in a family, and presence of consanguinity. Thus, we established whole exome sequencing as an efficient approach toward a molecular genetic diagnosis in individuals with nephrolithiasis/nephrocalcinosis who manifest before age 25 years.

Urinary stone disease (USD) is increasing in adult and pediatric populations. Adult and pediatric studies have demonstrated decreased bone mineral density and increased fracture rates. USD has also been independently linked to increased rates of myocardial infarction and cerebral vascular accidents. Although USD is a multisystem disorder involving the kidneys, bone, and vasculature, the molecular mechanisms linking these three organs remain unknown. Calcium oxalate nephropathy was induced in C57BL/6J mice with intra-peritoneal (ip) injection of sodium glyoxolate. Half of each kidney underwent Pizzalato staining and half was snap frozen for RNA extraction. RT2 Profiler Mouse Atherosclerosis, Osteoporosis, and Calcium Signaling PCR Arrays (Qiagen) were performed. Only results that passed quality checks in PCR array reproducibility and genomic DNA contamination were included. Genes had to show at least fourfold differential expression and P 10-fold increase. All 10 have P ≤ 0.003. The calcium signaling array showed significant fourfold upregulation of 10 genes, four of which were ≥10-fold. All 10 have P ≤ 0.03. We have demonstrated that calcium oxalate nephropathy can induce upregulation of atherosclerotic, metabolic bone, and calcium homeostasis genes in a murine model. This may be and initial step in identifying the molecular mechanisms linking stone, bone, and cardiovascular disease. J. Cell. Biochem. 118: 2744–2751, 2017. © 2017 Wiley Periodicals, Inc.

Kidney stones are increasing in the pediatric and adult populations; similarly osteoporosis is increasingly recognized in children. While kidney stone formers are known to suffer from low bone density, metabolic bone patients have not been considered a high risk population for kidney stones. Retrospective chart review of Nationwide Children’s Hospital Metabolic Bone Clinic patients from October 2009–2013. Patients were identified by ICD 9 codes for osteoporosis, osteopenia, low bone density and kidney stones. Only patients with radiologic evidence of both diseases were included.Twenty-six of 889 patients met criteria; this is equivalent to an incidence of 30 per 100,000 patients. Osteoporosis was the most frequent bone diagnosis. Males were the majority (68%). Most common secondary diagnoses: seizure (52%) and cerebral palsy (44%). Treatment: calcium (48%), vitamin D (40%), bisphosphonates (48%). The majority (75%) were non-ambulatory. Most frequent lithogenic medications: Topiramate (42%) and corticosteroids (27%). This is one of the first studies to consider metabolic bone patients as high risk for urinary stone disease. We found a higher rate of kidney stones in pediatric metabolic bone patients compared to data available for the general pediatric kidney stone population. The most common risk factor for bone and stone disease was nonambulatory status. Males were more frequently affected than females; this is the reverse of general adolescent kidney stone population. The predominance of cerebral palsy and seizure patients can be attributed to their frequency of non-ambulatory status and lithogenic medications such as Topiramate.

ObjectiveCurrent urinary tract infection (UTI) diagnostic strategies that rely on leukocyte esterase have limited accuracy. We performed an aptamer-based proteomics pilot study to identify urine protein levels that could differentiate a culture proven UTI from culture negative samples, regardless of pyuria status.MethodsWe analyzed urine from 16 children with UTIs, 8 children with culture negative pyuria and 8 children with negative urine culture and no pyuria. The urine levels of 1,310 proteins were quantified using the Somascan™ platform and normalized to urine creatinine. Machine learning with support vector machine (SVM)-based feature selection was performed to determine the combination of urine biomarkers that optimized diagnostic accuracy.ResultsEight candidate urine protein biomarkers met filtering criteria. B-cell lymphoma protein, C-X-C motif chemokine 6, C-X-C motif chemokine 13, cathepsin S, heat shock 70kDA protein 1A, mitogen activated protein kinase, protein E7 HPV18 and transgelin. AUCs ranged from 0.91 to 0.95. The best prediction was achieved by the SVMs with radial basis function kernel.ConclusionsBiomarkers panel can be identified by the emerging technologies of aptamer-based proteomics and machine learning that offer the potential to increase UTI diagnostic accuracy, thereby limiting unneeded antibiotics.

Autoantibodies targeting host antigens contribute to autoimmune disorders, frequently occur during and after infections and have been proposed to contribute to malaria-induced anemia. We measured anti-phosphatidylserine (PS) and anti-DNA antibody levels in 382 Ugandan children prospectively recruited in a study of severe malaria (SM). High antibody levels were defined as antibody levels greater than the mean plus 3 standard deviations of community children (CC). We observed increases in median levels of anti-PS and anti-DNA antibodies in children with SM compared to CC (p

Vesicoureteral reflux (VUR) is a complex, heritable disorder. Genome-wide linkage analyses of families affected by VUR have revealed multiple genomic loci linked to VUR. These loci normally harbor a number of genes whose biologically functional variant is yet to be identified. DNA copy number variations (CNVs) have not been extensively studied at high resolution in VUR patients. In this study, we performed array comparative genomic hybridization (aCGH) on a cohort of patients with a history of both VUR and urinary tract infection (UTI) with the objective of identifying genetic variations responsible for VUR and/or UTI susceptibility. UTI/VUR-associated CNVs were identified by aCGH results from the 192 Randomized Intervention for Children With Vesicoureteral Reflux (RIVUR) patients compared to 683 controls. Rare, large CNVs that are likely pathogenic and lead to VUR development were identified using stringent analysis criteria. Because UTI is a common affliction with multiple risk factors, we utilized standard analysis to identify potential disease-modifying CNVs that can contribute to UTI risk. Gene ontology analysis identified that CNVs in innate immunity and development genes were enriched in RIVUR patients. CNVs affecting innate immune genes may contribute to UTI susceptibility in VUR patients and may provide the first step in assisting clinical medicine in determining adverse outcome risk in children with VUR.

Background: Urinary stones are affecting more children, and pediatric stone formers have unique pathophysiology compared to adults. While adult stone formers are most frequently male, children have an age dependent sex prevalence. Under 10 years, a majority of stone formers are boys; adolescent stone formers are mostly female. Previous adult studies have shown that stone composition is influenced by the sex and age of the stone former. Thus, we hypothesize that female and male stone forming children will also have sex and age specific stone phenotypes.Methods: Retrospective chart review of a large pediatric center's stone forming children 6/1/2009 to 6/1/2016. Patients were identified by ICD 9 codes: N20, N20.1, and N20.9. Charts were reviewed for radiographic evidence of stones or documented visualized stone passage.Results: One hundred and thirty six subjects: 54 males and 82 females. Females were older, median age 14 years [interquartile range (IQR): 11, 15] vs. males' median age 12 years (IQR: 11, 14) (p < 0.01). Females had lower height z-scores, median 0.2 (IQR: −0.8, 0.8) vs. males' median 0.8 (IQR: −0.2, 1.8) (p < 0.01). Presenting symptoms were similar except flank pain affecting 39% of females vs. 22% of males (p = 0.04). Leukocyte esterase was positive in more females than males (33 vs. 4%) (p < 0.001). Males had a higher BUN/Cr ratio, mean ± standard deviation of 19.8 ± 6.3 vs. 16.6 ± 6.5 in females (p = 0.01). Glomerular hyperfiltration was present in 9% of patients while 35% of patients had estimated glomerular filtration rate (eGFR) < 90 ml/min/1.73 m2. Treatment strategies and clinical course were similar except females were told to increase dietary citrate more frequently than males (21 vs. 4%) (p < 0.01).Conclusion: We have provided a novel analysis and demonstrated that low height z-score and pyuria are more common in female stone formers. We have also shown that 9% of pediatric stone formers have labs consistent with hyperfiltration. Whether high protein intake and/or chronic dehydration are associated with hyperfiltration and long-term renal function in children with kidney stones will be an area for future research.

Expanding epidemiologic and physiologic data suggest that urinary stone disease is best conceptualized as a chronic metabolic condition punctuated by symptomatic, preventable stone events. These acute events herald substantial future chronic morbidity, including decreased bone mineral density, cardiovascular disease, and CKD. Urinary stone disease imposes a large and growing public health burden. In the United States, 1 in 11 individuals will experience a urinary stone in their lifetime. Given this high incidence and prevalence, urinary stone disease is one of the most expensive urologic conditions, with health care charges exceeding $10 billion annually. Patient care focuses on management of symptomatic stones rather than prevention; after three decades of innovation, procedural interventions are almost exclusively minimally invasive or noninvasive, and mortality is rare. Despite these advances, the prevalence of stone disease has nearly doubled over the past 15 years, likely secondary to dietary and health trends. The NIDDK recently convened a symposium to assess knowledge and treatment gaps to inform future urinary stone disease research. Reducing the public health burden of urinary stone disease will require key advances in understanding environmental, genetic, and other individual disease determinants; improving secondary prevention; and optimal population health strategies in an increasingly cost–conscious care environment.

The most common acute glomerulonephritis in children is poststreptococcal glomerulonephritis (PSGN) usually occurring between 3 and 12 years old. Hypertension and gross hematuria are common presenting symptoms. Most PSGN patients do not experience complications, but rapidly progressive glomerulonephritis and hypertensive encephalopathy have been reported. This paper reports 17 patients seen in 1 year for PSGN including 4 with atypical PSGN, at a pediatric tertiary care center. Seventeen children (11 males), mean age of 8 years, were analyzed. Ninety-four percent had elevated serum BUN levels and decreased GFR. Four of the hospitalized patients had complex presentations that included AKI along with positive ANA or ANCAs. Three patients required renal replacement therapy and two were thrombocytopenic. PSGN usually does not occur as a severe nephritis. Over the 12-month study period, 17 cases associated with low serum albumin in 53%, acute kidney injury in 94%, and thrombocytopenia in 18% were treated. The presentation of PSGN may be severe and in a small subset have associations similar to SLE nephritis findings including AKI, positive ANA, and hematological anomalies.

This case series describes subclinical development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in some patients and health care workers in a pediatric dialysis unit after contact with a seropositive patient.

BACKGROUND: Urinary stone are increasing in children, primarily during adolescence. Although urinary stones are often viewed in the context of intermittent stone events, increasing evidence indicates that stones are a metabolic process associated with chronic kidney disease and low bone mass. These aforementioned stone associated conditions may have pediatric origins. OBJECTIVE: To compare urine inflammatory markers in otherwise healthy stone forming children versus matched controls. METHODS: Urine samples were collected from 12 adolescents with urinary stones along with 15 controls. The levels of 30 urine cytokines were measured using a Mesoscale 30-Plex Human Cytokine panel and normalized to urine creatinine levels. RESULTS: Macrophage inflammatory protein 1β and Interleukin 13 levels were significantly elevated in the urine of the stone forming adolescents compared to controls. Interleukin 17A was elevated in the urine of controls. CONCLUSIONS: This study indicates that urine levels of cytokines involved in chronic inflammation and fibrosis are elevated urinary stone formers as early as adolescence. Because stone formers are at risk for chronic kidney disease, Macrophage inflammatory protein 1β and Interleukin 13 represent investigative targets.

Objectives: Low hydration has a deleterious effect on many conditions. In the absence of a urine concentrating defect, urine concentration is a marker of hydration status. However, markers to evaluate hydration status have not been well studied in children. The objectives of this paper are to compare measures of thirst and urine concentration in children and to develop a novel mobile technology application to measure urine concentration.Study Design: Children age 12–17 years were selected (n = 21) for this pilot study. Thirst perception, specific gravity (automated dipstick analysis and refractometer), and urine color scale results were correlated to urine osmolality. The technology department developed a mobile technology camera application to measure light penetrance into urine which was tested on 25 random anonymized urine samples.Results: The patients' thirst perception and color scale as well as two researchers color scale did not significantly correlate with osmolality. Correlation between osmolality and hydration markers resulted in the following Pearson coefficients: SG automated dipstick, 0.61 (P 0.003); SG refractometer, 0.98 (P < 0.0001); urine color scale (patient), 0.37 (P 0.10), and light penetrance, −0.77 (P < 0.0001). The correlation of light penetrance with osmolality was stronger than all measures except SG by refractometer and osmolality.Conclusion: The mobile technology application may be a more accurate tool for urine concentration measurement than specific gravity by automated dipstick, subjective thirst, and urine color scale, but lags behind specific gravity measured by refractometer. The mobile technology application is a step toward patient oriented hydration strategies.

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.

OBJECTIVES The aim of this study was to examine computed tomography (CT) and ultrasound (US) utilization trends in incident and prevalent pediatric emergency department (ED) urolithiasis patients before and after imaging guideline release. METHODS We reviewed imaging modalities for children with 2 or more ED encounters between January 1, 2006, and September 1, 2013, for urolithiasis using the Pediatric Health Information System database. Z scores compared the proportion of patient encounters receiving CT and US before (January 1, 2006, to December 31, 2010) and after (January 1, 2011, to September 1, 2013) the release of imaging guidelines. McNemar test for paired proportions compared the percentage of US and CT use between initial versus subsequent visits. Piecewise logistic regression was used to determine the probability of US use and CT use over time before and after the implementation of imaging guidance. RESULTS Analysis was completed on 2041 patients with 4930 unique encounters for urolithiasis. During 1758 encounters (35.7%), CT was performed initially. Ultrasound was performed 1585 times (32.2%). Fourteen percent fewer CT procedures were performed during first urolithiasis visits after guideline release (P < 0.01), whereas US use increased by 15% (P < 0.01). Fewer CT procedures were performed at later visits compared with the first (P < 0.05), and US was used more during second or later visits than the first (P < 0.05). CONCLUSIONS Medical providers at large academic pediatric EDs have decreased use of CT and increased use of US over the study time frame to diagnose urolithiasis and are now similar during initial visits (US 36.4% vs CT 36.2%, P = 0.94). Physicians are still more likely to use US as the initial urolithiasis imaging modality during second and later encounters.

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.

Aim To find the trend in patient’s visits to our centers for vesicoureteral reflux (VUR). We hypothesize that VUR diagnosis and hence possible nephropathy recognition may be diminishing because of changing practice patterns. Methods Data were extracted from electronic medical records for new and follow-up patients aged 0–18 years with ICD-9/10 codes to correspond with VUR, VUR unilateral, VUR bilateral, and VUR with reflux nephropathy, as well as new patients with diagnoses of urinary tract infections (UTI) and pyelonephritis at two major pediatric centers from 2012 to 2015. Figures and statistics to reflect absolute clinic visits and annual trends were created with SPSS 2010. Linear regression was applied. Results Annually, Le Bonheur Children’s Hospital and Nationwide Children’s Hospital experienced an average decrease of 13 and 17% in total VUR visits, and an average decrease of 22 and 27% in VUR nephropathy visits, respectively, for each institution. Patient visits for UTIs were reduced an average of 16% annually in both centers. Linear regression demonstrated that number of patients (patients/year ± SE) decreased annually 69 ± 19 (P = 0.02), 7 ± 2 (P = 0.02), and 67 ± 25 (P = 0.04) for VUR, VUR nephropathy, and UTI, respectively. Conclusion We conclude that the decreased number of VUR and VUR nephropathy cases identified in subspecialty clinics (Nephrology/Urology) at two major children’s hospitals reflect a possible decreased identification of VUR. This trend may also be due to decreased referral of low grade cases of VUR. We cannot conclude that “undifferentiated UTI” referrals increased concomitantly to account for the decreased VUR as our data reflects a decreased trend in those visits as well. We suggest that clinicians following the American Academy of Pediatrics guidelines ensure that all UTI are accounted for and surveillance is appropriately escalated for recurrent UTI or abnormal imaging results.

Recent studies stress the importance of antimicrobial peptides in protecting the urinary tract from infection. Previously, we have shown that ribonuclease 7 (RNase 7) is a potent antimicrobial peptide that has a broad-spectrum antimicrobial activity against uropathogenic bacteria. The urothelium of the lower urinary tract and intercalated cells of the kidney produce RNase 7, but regulation of its antimicrobial activity has not been well defined. Here, we characterize the expression of an endogenous inhibitor, ribonuclease inhibitor (RI), in the urinary tract and evaluate its effect on the antimicrobial activity of RNase 7. Using RNA isolated from non-infected human bladder and kidney tissue, quantitative real-time polymerase chain reaction showed that RNH1, the gene encoding RI, is constitutively expressed throughout the urinary tract. With pyelonephritis, RNH1 expression and RI peptide production significantly decrease. Immunostaining localized RI production to the umbrella cells of the bladder and intercalated cells of the renal collecting tubule. In vitro assays showed that RI bound to RNase 7 and suppressed its antimicrobial activity by blocking its ability to bind the cell wall of uropathogenic bacteria. Thus, these results demonstrate a new immunomodulatory role for RI and identified a unique regulatory pathway that may affect how RNase 7 maintains urinary tract sterility.

Intercalated cells of the collecting duct (CD) are critical for acid-base homeostasis and innate immune defense of the kidney. Little is known about the impact of acidosis on innate immune defense in the distal nephron. Urinary tract infections are mainly due to Escherichia coli and are an important risk factor for development of chronic kidney disease. While the effect of urinary pH on growth of E. coli is well established, in this study, we demonstrate that acidosis increases urine antimicrobial activity due, at least in part, to induction of cathelicidin expression within the CD. Acidosis was induced in rabbits by adding NH4Cl to the drinking water and reducing food intake over 3 days or by casein supplementation. Microdissected CDs were examined for cathelicidin mRNA expression and antimicrobial activity, and cathelicidin protein levels in rabbit urine were measured. Cathelicidin expression in CD cells was detected in kidney sections. CDs from acidotic rabbits expressed three times more cathelicidin mRNA than those isolated from normal rabbits. Urine from acidotic rabbits had significantly more antimicrobial activity (vs. E. coli) than normal urine, and most of this increased activity was blocked by cathelicidin antibody. The antibody had little effect on antimicrobial activity of normal urine. Urine from acidotic rabbits had at least twice the amount of cathelicidin protein as did normal urine. We conclude that metabolic acidosis not only stimulates CD acid secretion but also induces expression of cathelicidin and, thereby, enhances innate immune defense against urinary tract infections via induction of antimicrobial peptide expression.

The mechanisms that maintain sterility in the urinary tract are incompletely understood; however, recent studies stress the importance of antimicrobial peptides in protecting the urinary tract from infection. Ribonuclease 7 (RNase 7), a potent antimicrobial peptide contributing to urinary tract sterility, is expressed by intercalated cells in the renal collecting tubules and is present in the urine at levels sufficient to kill bacteria at baseline. Here, we characterize the expression and function of RNase 7 in the human urinary tract during infection. Both quantitative real-time PCR and enzyme-linked immunosorbant assays demonstrated increases in RNASE7 expression in the kidney along with kidney and urinary RNase 7 peptide concentrations with infection. While immunostaining localized RNase 7 production to the intercalated cells of the collecting tubule during sterility, its expression during pyelonephritis was found to increase throughout the nephron but not in glomeruli or the interstitium. Recombinant RNase 7 exhibited antimicrobial activity against uropathogens at low micromolar concentrations by disrupting the microbial membrane as determined by atomic force microscopy. Thus, RNase 7 expression is increased in the urinary tract with infection and has antibacterial activity against uropathogens at micromolar concentrations.

Acquired renal scarring occurs in a subset of patients following febrile urinary tract infections and is associated with hypertension, proteinuria, and chronic kidney disease. Limited knowledge of histopathology, immune cell recruitment, and gene expression changes during pyelonephritis restricts the development of therapies to limit renal scarring. Here, we address this knowledge gap using immunocompetent mice with vesicoureteral reflux. Transurethral inoculation of uropathogenic Escherichia coli in C3H/HeOuJ mice leads to renal mucosal injury, tubulointerstitial nephritis, and cortical fibrosis. The extent of fibrosis correlates most significantly with inflammation at 7 and 28 days postinfection. The recruitment of neutrophils and inflammatory macrophages to infected kidneys is proportional to renal bacterial burden. Transcriptome analysis reveals molecular signatures associated with renal ischemia-reperfusion injury, immune cell chemotaxis, and leukocyte activation. This murine model recapitulates the cardinal histopathological features observed in humans with acquired renal scarring following pyelonephritis. The integration of histopathology, quantification of cellular immune influx, and unbiased transcriptional profiling begins to define potential mechanisms of tissue injury during pyelonephritis in the context of an intact immune response. The clear relationship between inflammatory cell recruitment and fibrosis supports the hypothesis that acquired renal scarring arises as a consequence of excessive host inflammation and suggests that immunomodulatory therapies should be investigated to reduce renal scarring in patients with pyelonephritis.

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

Calcium nephrolithiasis in children is increasing in prevalence and tends to be recurrent. Although children have a lower incidence of nephrolithiasis than adults, its etiology in children is less well understood; hence treatments targeted for adults may not be optimal in children. To better understand metabolic abnormalities in stone forming children, we compared chemical measurements and the crystallization properties of 24-hour urine collections from 129 stone formers matched to 105 non-stone forming siblings and 183 normal, healthy children with no family history of stones; all aged 6 to 17 years. The principal risk factor for calcium stone formation was hypercalciuria. Stone formers have strikingly higher calcium excretion along with high supersaturation for calcium oxalate and calcium phosphate, and a reduced distance between the upper limit of metastability and supersaturation for calcium phosphate, indicating increased risk of calcium phosphate crystallization. Other differences in urine chemistry that exist between adult stone formers and normal individuals such as hyperoxaluria, hypocitraturia, abnormal urine pH and low urine volume were not found in these children. Hence, hypercalciuria and a reduction in the gap between calcium phosphate upper limit of metastability and supersaturation are crucial determinants of stone risk. This highlights the importance of managing hypercalciuria in children with calcium stones.

Although the urinary tract is constantly challenged by microbial invasion, it remains free from colonization. Although little is known about how the urinary tract maintains sterility, the presence of antimicrobial peptides (AMPs) in the urine suggests that they may play a role in its protection from infection. Ribonuclease 7 (RNase 7) is a potent AMP that was first identified in the skin. Here, we characterize the expression and relevance of RNase 7 in the human kidney and urinary tract. Using RNA isolated from healthy human tissue, we performed quantitative real-time PCR and found basal RNASE7 expression in kidney and bladder tissue. Immunohistochemical and immunofluorescent analysis localized RNase 7 to the urothelium of the bladder, ureter, and the intercalated cells of the collecting tubules. In control urine samples from healthy individuals, the concentration of RNase 7 was found to be in the low micromolar range; very abundant for an AMP. Antibacterial neutralization assays showed that urinary RNase 7 has potent antimicrobial properties against Gram-negative and Gram-positive uropathogenic bacteria. Thus, RNase 7 is expressed in the human kidney and urinary tract and it may have an important antimicrobial role in maintaining tract sterility.

Summary Purpose The metabolic evaluation of children with nephrolithiasis begins with a 24-h urine collection. For adults, the diagnostic yield increases with consecutive collections; however, little is known regarding the variability of multiple 24-h studies in the pediatric population. We sought to evaluate the variability of consecutive 24-h urine collection in children through a multi-institutional study hypothesizing that compared with a single collection, consecutive 24-h urine collections would reveal a greater degree of clinically useful information in the evaluation of children at risk for nephrolithiasis. Materials and methods Including data from six institutions, we identified children less than 18 years of age considered at risk for recurrent nephrolithiasis, undergoing metabolic evaluation. We evaluated a subset of patients performing two collections with urine creatinine varying by 10% or less during a 7-day period. Discordance between repeat collections based on normative urine chemistry values was evaluated. Results A total of 733 children met inclusion criteria, and in over a third both urine calcium and urine volume differed by 30% or more between samples. Urine oxalate demonstrated greater variation between collections in children p = 0.030) while variation in other parameters did not differ by age. Discordance between repeat samples based on normative values was most common for urine oxalate (22.5%) and the derived relative supersaturation ratios for both calcium phosphate (25.1%) and calcium oxalate (20.5%). The proportion of discordant samples, based on normative thresholds, as well as variability greater ≥30% and 50%, respectively, are shown in the table. Conclusions Our analysis indicates that stone risk in as many as one in four children may be misclassified if normative values of only a single 24-h urine are used. In light of these findings, repeat 24-h urine collections prior to targeted intervention to modify stone risk are advised to increase diagnostic yield in children at risk for nephrolithiasis. Table . Percent variability and discordant samples (based on normative thresholds) for urinary parameters in consecutive 24-h urine studies in children. Parameter Percent variability Discordant samples ≥30% ≥50% Calcium ( N , %) 219 (34.2) 102 (15.9) 88 (13.1) Oxalate ( N , %) 94 (23.3) 28 (6.9) 104 (22.5) Citrate ( N , %) 92 (16.6) 22 (4.0) 42 (8.2) SS CaOx ( N , %) N/A N/A 138 (20.5) SS CaPhos ( N , %) N/A N/A 169 (25.1) Volume 269 (36.8) 89 (12.2) N/A

Urinary tract infections (UTI) are a common condition in children. Vesicoureteral reflux (VUR) represents a common associated condition with childhood UTI. UTI susceptibility appears to have a genetic component based on family and UTI cohort studies. Targeted analysis of innate immune system genetic variations indicate that these variations are important in UTI susceptibility. In this overview, we discuss how current cohorts and genetic strategies can be implemented to discover new susceptibility loci in patients with UTI.

Urinary stone disease (USD) is an increasing clinical problem in both children and adults. One in ten individuals will experience a urinary stone, yet the mechanisms responsible for urinary stones remain largely unknown. Bacteria have long been recognized to contribute to struvite urinary stones; however, the role of bacteria in the development of the more common calcium oxalate (CaOx) and calcium phosphate (CaPhos) stones has not been extensively investigated. However, several findings do indicate a possible association between urinary stones and bacteria, including the high rate of urinary tract infections (UTI) in urinary stone patients and multiple case series of culture-positive urinary stones, including stones composed of CaOx or CaPhos. New technology, such as next generation sequencing, may be used to lend additional insight regarding the association between urinary stones and bacteria. In 2015, we published the initial bacterial sequencing results from five urinary stones, from which we sequenced multiple types of bacterial DNA. Whether these bacteria are causal, disease modifying or passively present remains to be determined. However, initial exploration of underlying mechanisms for this association indicate that bacteria aggregate selectively to crystals, that their presence is associated with increased clumping of crystals, and that they stimulate incorporation of proteins into the stone matrix.

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.

A novel COL4A5 mutation causes rapid progression to end-stage renal disease in males, despite the absence of clinical and biopsy findings associated with Alport syndrome. Affected males have proteinuria, variable hematuria, and an early progression to end-stage renal disease. Renal biopsy findings include global and segmental glomerulosclerosis, mesangial hypercellularity and basement membrane immune complex deposition. Exon sequencing of the COL4A5 locus identified a thymine to guanine transversion at nucleotide 665, resulting in a phenylalanine to cysteine missense mutation at codon 222. The phenylalanine at position 222 is absolutely conserved among vertebrates. This mutation was confirmed in 4 affected males and 4 female obligate carriers, but was absent in 6 asymptomatic male family members and 198 unrelated individuals. Immunostaining for α5(IV) collagen in renal biopsies from affected males was normal. This mutation, in a non-collagenous interruption associated with severe renal disease, provides evidence for the importance of this structural motif and suggests the range of phenotypes associated with COL4A5 mutations is more diverse than previously realized. Hence, COL4A5 mutation analysis should be considered when glomerulonephritis presents in an X-linked inheritance pattern, even with a presentation distinct from Alport syndrome.