Defensin–Encoding DEFA1A3 Associate with Urinary Tract Infection Risk in Children with Vesicoureteral Reﬂux

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Polymorphisms in a-Defensin–Encoding DEFA1A3 Associate with Urinary Tract Infection Risk in Children with Vesicoureteral Reﬂux

† ‡ Andrew L. Schwaderer,* Huanyu Wang,* SungHwan Kim, Jennifer M. Kline, Dong Liang,§ | † Pat D. Brophy, Kirk M. McHugh,¶ George C. Tseng, Vijay Saxena,* Evan Barr-Beare,* †† ‡ Keith R. Pierce,§ Nader Shaikh,** J. Robert Manak, Daniel M. Cohen, Brian Becknell,* ‡‡ || John D. Spencer,* Peter B. Baker, Chack-Yung Yu,§§ and David S. Hains§

*The Centers for Clinical and Translational Medicine and §§Molecular and Human Genetics, The Research Institute at Nationwide Children’s Hospital, Columbus, Ohio; Departments of †Biostatistics and **Pediatrics, University of Pittsburgh, Pittsburgh, Pennsylvania; ‡Emergency Medicine and ‡‡Department of Pathology, Nationwide Children’s Hospital, Columbus, Ohio; §Innate Immunity Translational Research Center, Children’s Foundation Research Institute at Le Bonheur Children’s Hospital, Memphis, Tennessee; |Division of Nephrology, Department of Pediatrics, University of Iowa Children’s Hospital, Iowa City, Iowa; ¶Division of Anatomy, The Ohio State University, Columbus, Ohio; ††Departments of Biology and Pediatrics, University of Iowa, Iowa; and ||Department of Pediatrics, University of Tennessee Health Science Center, Memphis, Tennessee

ABSTRACT The contribution of genetic variation to urinary tract infection (UTI) risk in children with vesicoureteral reflux is largely unknown. The innate immune system, which includes antimicrobial peptides, such as the a-defensins, encoded by DEFA1A3, is important in preventing UTIs but has not been investigated in the vesicoureteral reflux population. We used quantitative real–time PCR to determine DEFA1A3 DNA copy numbers in 298 individuals with confirmed UTIs and vesicoureteral reflux from the Randomized Interven- tion for Children with Vesicoureteral Reflux (RIVUR) Study and 295 controls, and we correlated copy numbers with outcomes. Outcomes studied included reflux grade, UTIs during the study on placebo or antibiotics, bowel and bladder dysfunction, and renal scarring. Overall, 29% of patients and 16% of controls had less than or equal to five copies of DEFA1A3 (odds ratio, 2.09; 95% confidence interval, 1.40 to 3.11; P,0.001). For each additional copy of DEFA1A3, the odds of recurrent UTI in patients receiving antibiotic prophylaxis decreased by 47% when adjusting for vesicoureteral reflux grade and bowel and bladder dysfunction. In patients receiving placebo, DEFA1A3 copy number did not associate with risk of recurrent UTI. Notably, we found that DEFA1A3 is expressed in renal epithelium and not restricted to myeloid-derived cells, such as neutrophils. In conclusion, low DEFA1A3 copy number associated with recurrent UTIs in subjects in the RIVUR Study randomized to prophylactic antibiotics, providing evidence that copy number polymorphisms in an antimicrobial peptide associate with UTI risk.

J Am Soc Nephrol 27: 3175–3186, 2016. doi: 10.1681/ASN.2015060700

Vesicoureteral reﬂux (VUR), which is present in Received June 25, 2015. Accepted January 13, 2016. – 1% 2% of children, is a risk factor for the develop- A.L.S. and H.W. contributed equally to this work. ment of urinary tract infections (UTIs).1 The Ran- Published online ahead of print. Publication date available at domized Intervention for Children with Vesicoureteral www.jasn.org. Reﬂux (RIVUR) Trial recently randomized children Correspondence: Dr. David S. Hains, Children’sFoundation with a history of UTI and VUR to daily antibiotic Research Institute, Innate Immunity Translational Research Center, 50 prophylaxis or placebo and followed them for 2 years North Dunlap Street, Memphis, TN, 38103. Email: [email protected] to monitor for UTI recurrence risk. Additional details Copyright © 2016 by the American Society of Nephrology

J Am Soc Nephrol 27: 3175–3186, 2016 ISSN : 1046-6673/2710-3175 3175 CLINICAL RESEARCH www.jasn.org about the RIVUR Study design and outcomes have been pre- DEFA1A3 DNA copy numbers, contribute to UTI risk in pa- viously reported as well as our methods.2–4 The RIVUR Study tients with VUR. The objectives of this study included (1) showed that 25.4% of children with VUR treated with placebo comparison of DEFA1A3 CNP in children from the RIVUR experience UTI recurrence over a 2-year period, whereas anti- Trial who had a history of VUR and one or two UTIs with biotic prophylaxis reduces the UTI recurrence risk to 13%.3 healthy controls and (2) determination of the biologic rele- These recurrent UTIs can lead to renal scarring, hypertension, vance of DEFA1A3 expression in the kidney.2,3 and CKD.5,6 However, the broad application of antibiotic prophylaxis to all patients with VUR remains controversial, because only 30% of children with VUR will experience an initial UTI.7,8 RESULTS Therefore, strategies are needed to identify a subset of patients with VUR who will experience UTIs and benefit from antibiotic Patient Cohorts Analyzed Were Matched for Sex and prophylaxis. Ethnic/Racial Category The innate immune system is largely responsible for the DNA was obtained on 298 white girl patients in the RIVUR body’s defense against UTIs. Innate immune effectors include Trial. Two hundred ninety-five white girl controls with no antimicrobial peptides (AMPs) that rapidly destroy microbes history of UTI or VUR were enrolled between 2010 and 2014. and chemokines that result in inflammatory cell migration to The mean age of the patients in the RIVUR Trial was 22.7 the affected site and the inflammatory cells.9–11 AMPs, such as months (range =2–71) at the start of the 2-year study. defensins, have a broad range of antimicrobial activity The mean age of the control patients was 129.8 months against a wide variety of microbes, including Gram-negative (range =3.5–215). The patients in the RIVUR Trial were and -positive bacteria.12 Defensins are categorized into two matched with controls the same age or older to have equal or families, the a-defensins and the b-defensins, depending on more years to acquire a UTI. Complete clinical characteristics their disulfide-bridging pattern. b-Defensins are widely ex- of our cohorts are summarized in Table 1. pressed by epithelial cells, whereas a-defensins are expressed by neutrophils or in the case of a-defensin-5 (DEFA5), the Patients in the RIVUR Study Have Decreased DNA epithelia of the gastrointestinal tract, the genitourinary tract Copy Number of DEFA1A3 Compared with Controls in girls, and the urinary tract.13–15 We determined DEFA1A3 gene copy number per diploid ge- Neutrophil-associated DEFA1 differs from an adjacent nome using quantitative real–time PCR and verified it in a gene, DEFA3, by a single nucleotide. This gene locus has col- subset of samples using pulsed–field gel electrophoresis. Pa- lectively been renamed DEFA1A3, and it encodes the protein tients in the RIVUR Trial and matched controls were copy Human Neutrophil Peptide 1–3(HNP1–3).16 Historically, typed. The DEFA1A3 copy number in the patients in the myeloid-derived cells were the only cells thought to produce RIVUR Trial varied from three to 13, with a mean copy num- HNP1–3.17–19 HNP1–3 possesses broad spectrum antimicro- ber of 6.361.4. The DEFA1A3 copy number for controls bial activity against a range of bacteria, including Escherichia ranged between four and 18, with a mean copy number 762.0. coli, and urine levels of HNP1–3increaseeightfoldwith The distributions of copy numbers were significantly pyelonephritis versus controls.18,20,21 However, DEFA1A3/ different between the two groups (P,0.001) (Figure 1). HNP1–3 has not been extensively evaluated in the kidney. Overall, a higher percentage of patients in the RIVUR Trial Genetic variations in humans range from single-nucleotide (n=86; 29%) had five or fewer copies of DEFA1A3 compared changesto large microscopically visible chromosome defects.22 with controls (n=48; 16%; odds ratio [OR], 2.09; 95% confi- Duplications, deletions, or insertions of $1000 bp have been dence interval, 1.40 to 3.11; P,0.001). Conversely, a higher identified in humans and designated DNA copy number var- percentage of controls (n=93; 31%) had eight or more copies iations (CNVs).23 Some genes, including DEFA1A3, exist as compared with patients in the RIVUR Trial (n=55; 18%; OR, multiallelic genes, meaning that they exist in multiple copies 0.50; 95% confidence interval, 0.34–0.73); P,0.001). A similar (more than two per diploid genome). Because 70% of CNVs statistically significant difference was seen in white boys in span genes, they likely affect genetic networks. Thus, although single-nucleotide polymorphisms and point mutations occur Table 1. Clinical characteristics of subjects and controls more frequently, CNVs are more likely to be important com- Characteristics RIVUR, N=298 Control, N=295 ponents of genomic diversity and phenotypic variation by – – their ability to generate significant gene dosage effects.24 Mean age, mo (range) 22.7 (2 71) 129.8 (3.5 215) White, % 100 100 CNVs that occur in .1% of the population have been Girls, % 100 100 termed copy number polymorphisms (CNPs). CNPs may rep- Antibiotic group, n (%) 154 (52) N/A fi resent excellent candidates for disease risk modi ers. For in- Breakthrough UTI 23 (15) N/A stance, DEFA1A3 CNPs have been associated with various Placebo group, n (%) 144 (48) N/A inflammation and autoimmune diseases, including sepsis, Breakthrough UTI 47 (33) N/A Crohn disease, and Behcet disease.25–27 We hypothesize that Acquired scar, n (%) 22 (9) N/A deficiencies in the host innate immune defense, such as low N/A, not applicable.

3176 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 3175–3186, 2016 www.jasn.org CLINICAL RESEARCH the RIVUR Trial compared with control white boys (Supple- group. Patients treated with antibiotic prophylaxis who expe- mental Figure 1). Thus, a lower copy number of DEFA1A3 was rienced breakthrough UTIs (5.82 copies) averaged a 0.5-copy found in patients in the RIVUR Trial (VUR and UTI). difference compared with those without breakthrough UTIs (6.34 copies; n=131). These results were statistically signifi- Certain Patients in the RIVUR Study with Breakthrough cant (P=0.04). Patients treated with placebo who experienced Infections Have a Significantly Lower Copy Number of breakthrough infections (n=47) averaged 6.36 copies, similar DEFA1A3 to 6.39 copies in those who did not (n=97; P=0.90). This To determine if DEFA1A3 copy numbers were associated finding indicates that antibiotics in conjunction with a high with subsequent infections in the RIVUR Trial population (greater than or equal to eight) copy number of DEFA1A3 may during the study, we analyzed the relation of DEFA1A3 copy decrease UTI risk in children with VUR (OR, 0; 95% confi- number to UTI breakthrough events within the RIVUR Trial dence interval, 0-NaN; P=0.01) (Figure 2, B–D). Conversely, cohort. The distributions of DEFA1A3 copy number in those within the antibiotic group, low (less than or equal to five) with breakthrough infections (n=70) during the 2-year clin- DEFA1A3 copy number may increase UTI risk (OR, 2.51; 95% ical study compared with those who did not have break- confidence interval, 1.02 to 6.22; P=0.04). through UTIs (n=228) were not statistically different Because grade of VUR and bowel and bladder dysfunction (P=0.39). (BBD) have been identified as risk factors for UTIin the RIVUR The RIVUR Study was a placebo–controlled, randomized Trial cohort, we performed logistical regression analysis to trial that showed that daily antibiotic prophylaxis significantly account for these conditions (Table 2).4 Our analysis showed reduced UTI risk in patients.3 To explore the relations of CNV that DEFA1A3 copy number is a risk factor for UTI indepen- to recurrent UTI, we analyzed copy number in those with and dent of BBD or high-grade/dilating VUR in the antibiotic pro- without breakthrough UTIs in the antibiotic prophylaxis phylaxis group. For each additional copy of DEFA1A3,the 2 2 (n=154) and placebo (n=144) groups separately (Figure 2). odds of recurrent UTI decrease by 47% [1 ( 0.6307)3100%] For our study cohort, breakthrough infections occurred in 23 when adjusting for VUR grade, BBD, treatment group, and (15%) in the antibiotic group and 47 (33%) in the placebo interaction with copy number (Table 2). Additional analyses

Figure 1. Patients in the RIVUR Trial are more likely to have low DEFA1A3 copy number than control patients. (A) Bar graph representation of absolute DEFA1A3 copy number distribution between patients in the RIVUR Trial and control patients. (B) Bar graph representation of patients in the RIVUR Trial and control populations grouped according to low (less than or equal to ﬁve copies), midrange (six or seven copies), and high (greater than or equal to eight copies) number of DEFA1A3. (C) The ORs for patients in the RIVUR Trial cohort compared with controls and risk of having a UTI according to low, midrange, and high DEFA1A3 copy number. Patients in the RIVUR Trial have the additional risk factor of VUR, whereas controls do not.

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Figure 2. DEFA1A3 copy number predicts antibiotic response. (A and B) When analysis for breakthrough UTI is restricted to patients on antibiotic prophylaxis, a lower absolute DNA copy number significantly correlates with UTI recurrence (P=0.04). (C) In patients who received prophylaxis, the breakthrough UTI ORs for greater than or equal to eight, six or seven, and less than or equal to five copy number were 0.08, 0.98, and 2.51, respectively. (D and E) No difference was shown between DEFA1A3 copy number and recurrence in patients who received placebo (P=0.90). (F) The breakthrough UTI ORs were 0.70, 0.52, and 0.71 for greater than or equal to eight, six, or seven, and less than or equal to five copy number, respectively.

3178 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 3175–3186, 2016 www.jasn.org CLINICAL RESEARCH

Table 2. Logistical regression coefficients for recurrent UTI The mRNA Level of DEFA1A3 in Kidney Is Correlated risk among patients in the RIVUR Trial with the Gene Copy Number All Patients in the RIVUR Trial Because significant differences in gene copy number could Variable P result in large ranges of tissue mRNA expression, we correlated Estimate SEM Value tissue DNA copy number of DEFA1A3 to tissue DEFA1A3 28 Intercept 2.029 1.558 0.19 mRNA. Analyzing 43 kidney samples, we observed a positive DEFA1A3 CN 20.631 0.265 0.02 correlation between gene copy number and mRNA levels 2 BBD 0.373 0.339 0.27 (r =0.11; P=0.03) (Figure 5). VUR gradea 21.57 0.328 0.63 Group (P)b 23.087 1.7759 0.08 Interaction of 0.637 2.99 0.03 DISCUSSION DEFA1A3 CN 3 group (P) In this study, we used a unique and well characterized cohort VUR divided into nondilating (grades 1 and 2) versus dilating. CN, copy from the previously published RIVUR Study to show that number; P, placebo group. aVUR graded by grade 1 and 2 versus grades 3 and 4. patients with VUR and UTIs have a higher frequency of low bTreatment group (antibiotic prophylaxis group versus placebo). DEFA1A3 DNA copies compared with controls.2,29 Further- more, we have shown that the protein product, HNP1–3, is were done for each condition individually, and similar trends/ also produced by renal tubular epithelium. Finally, we show odds were seen (data not shown). that, despite the fact that DEFA1A3/HNP1–3 kidney expression was limited to the collecting duct, we can correlate host DEFA1A3 Copy Number May Confer a Risk to DNA copy number of DEFA1A3 to mRNA transcript levels in Nonclassic UTIs the kidney. Each UTI during the RIVUR Study had urine cultured and The role of the innate immune system and AMPs in kidney bacteria speciated. We analyzed DEFA1A3 copy number in disease and infections of the kidney and urinary tract is an relation to the bacteria type. We grouped patients as E. coli emerging concept. Our research group has shown the biologic (n=267) versus non–E. coli (n=31) UTI. The non–E. coli group relevance of AMPs ribonucleases 6 and 7 and human DEFA5 in had lower copy number compared with those with classic the kidney and urinary tract in relation to UTIs.14,30,31 Prior E. coli UTI (P=0.05) (Supplemental Figure 2). studies have shown additional AMPs that may be important to the innate defense of the kidney and urinary tract.13,32–34 Now, DEFA1A3 Copy Number Was Not Associated with HNP1–3 and its gene DEFA1A3 can be added to the growing Acquired Renal Scarring in the RIVUR Trial Cohort portfolio of AMPs that are critical to the innate defense of Within the analyzed patients in the RIVURTrial, 22of 240 (9%) kidney. Furthermore, the location of DEFA1A3 expression in acquired a new renal scar during the study period. DEFA1A3 the medullary collecting duct adds to the growing body of copy number did not associate with these new scars (Supple- evidence that the collecting duct, specifically intercalated cells, mental Figure 3). is critical to defending the kidney from ascending infection.35,36 Additionally, this study represents the first report DEFA1A3 mRNA Transcripts and HNP1–3 Are Present of genetic variations in an AMP gene being associated with in Collecting Duct Epithelium UTI risk. In healthy human kidney, DEFA1A3 mRNA expression ranged DEFA1A3 has previously been implicated in inflammatory from 15 to 3979 (median =399; n=37) transcripts per 10 ng renal diseases, including lupus nephritis and IgA nephropa- total RNA (Figure 3). Bladders had significantly lower levels, thy.37–39 Thus, DEFA1A3 CNPs may be a disease-modifying with a range of 11–91 (median =53; n=4) transcripts per 10 ng factor in a range of kidney diseases. Previous groups have total RNA (P,0.01). To confirm DEFA1A3 parenchymal ex- linked myeloid cell–derived HNP1–3 and genetic variations pression in the kidney, we performed in situ hybridization to in DEFA1A3 under the presumption that this gene is not ex- localize expression in the kidney (Figure 4A, Supplemental pressed by renal tissue. Our evidence that this gene is expressed Figure 4). Interestingly, DEFA1A3 transcripts localized to by renal epithelia opens many new avenues for research in the the epithelia of the nephron and were not present solely in fields of lupus nephritis and IgA nephropathy pathogenesis and resident neutrophils. To confirm translation of DEFA1A3 novel therapeutics. Strategies to determine the role of epithelial- mRNAtranscriptstothe protein HNP1–3 and localize neph- versus leukocyte-derived HNP1–3 will be important research ron segment, immunofluorescence was performed on endeavors. Although HNP1–3 has been associated with inflam- healthy human kidney. HNP1–3 localized to the medullary matory diseases, it is primarily an AMP. Previous research has collecting duct and was variably expressed by collecting duct identified its activity against E. coli.21 Our findings of increased intercalated and/or principal cells (Figure 4B), whereas no urinary levels during UTI as well as its location in the renal expression was present in negative control (Supplemental collecting duct and neutrophils conceptually align HNP1–3 Figure 5). with the bacterial defense of the kidneys and urinary

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Figure 3. DEFA1A3 is variably expressed in the kidney. DEFA1A3 is expressed in the kidney (n=37) in variable levels (mRNA copies per 10 ng total RNA), with a median of 399.0 and a range from 15.0 to 3973.0. The bladder (n=4) DEFA1A3 expression was minimal, with a median of 52.5 and a range from 11.0 to 91.0 (significantly less than the kidney; P,0.01; n=37 kidneys and n=4 bladders). tract.11,20,40 Thus, the link between genetic variations in this locus and UTIs is logical. CNVs and CNPs have been statistically associated with several disease phenotypes; however, the functional relevance of these genetic variations remains largely unknown.41,42 Here, we show that higher DEFA1A3 copy number results in increased mRNA expression, likely from increased gene dosage. The fact that prior analyses of DEFA1A3 copy number and expression levels in leukocytes did not show a correlation may be attributed to the observation that DEFA1A3 is largely transcribed and translated in bone marrow as opposed to cir- culating leukocytes.16,18,19 Therefore, decreased copy number in the patients in the RIVUR Trial theoretically results in decreased kidney expression of HNP1–3. Our findings present the kidney as an advantageous location for future studies eval- uating the correlation between gene copy number, protein expression, and disease risk. Our group and others have shown that urine levels of HNP1–3 markedly increase during UTI and pyelonephritis.11,20,40 We speculate that low HNP1–3 expression related to decreased DEFA1A3 copy number contributes to UTI risk on the basis of lower DEFA1A3 copy number in patients in the RIVUR Trial and the correlation between DEFA1A3 copy number and mRNA expression. To definitively show a causal relationship, future studies will need to measure urine HNP1– Figure 4. DEFA1A3 is expressed in the renal collecting duct. (A) 3 levels during UTI and DEFA1A3 copy number along with In situ hybridization for localization of DEFA1A3 in human kidney controlling for the amount of pyuria and the severity of UTI in sections. The hybridization of a DEFA1A3 antisense probe (pur- addition to renal epithelial versus tubular DEFA1A3 infection. ple staining) can be seen in isolated renal tubular epithelial cells Some functions of HNP1–3 have previously been shown to (arrows). (B) HNP1–3 kidney expression localizes to the medullary be dose dependent, such as activity against Mycobacterium collecting duct. HNP1–3 localization in the human kidney is tuberculosis, tissue remodeling properties, and inhibition of presented in (B) 340 and (C) 3100 images. Collecting ducts are adenovirus.43–45 Possibly, mechanisms other than copy number- labeled with aquaporin-2 (red), HNP1–3 is green, and nuclei are protein correlation are responsible for low DEFA1A3–associated blue. HNP1–3 immunolabeling (arrows) localized to the collecting UTI risk. tubules represented by aquaporin-2 staining (red, arrowheads) and isolated round polymorphonuclear cells consistent with Others have accurately copy-typed DEFA1A3 using PCR- – based methodologies and single-nucleotide polymorphism neutrophils (asterisks). Rare co-localization of HNP1 3and aquaporin-2 is noted (bolts). Scale bars, 50 mm. association with high/medium/low copy numbers.16,46 We

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Currently, many recommendations exist to limit unneces- sary or invasive testing to diagnose VUR or renal scarring. The AAP recommendations and the NICE guidelines help guide the clinician in determining which patients should be imaged with what imaging modality.47,48 Clear evidence–based recommendations are not available on how to treat the patient with known VUR, and this topic is a hot area of debate among clinical circles. Our study may provide the foundation for future studies to incorporate genetic data into clinical decision– making algorithms for patients with VUR at risk for recurrent UTIs. We acknowledge that this study has some limitations. Because the largest cohort from the RIVURTrial was white girls, our results may not apply to other cohorts and nonrefluxing Figure 5. DEFA1A3 mRNA expression correlates with copy patients. The DEFA1A3 copy number data from white boys also number. Regression analysis shows that increased DEFA1A3 gene copy number results in increased mRNA expression (black must be cautiously interpreted, because this comparison line). The 95% confidence intervals are represented by gray lines involved a small sample size. Future studies will focus on other (n=43 kidneys). cohorts with various UTI risk phenotypes and sex, race, and ethnicity. We have correlated DEFA1A3 copy number with kidney used multiple methodologies to accurately quantitate the ab- mRNA expression to complement a prior report of a positive solute copy number of DEFA1A3 in this study. The finding correlation with HNP1–3expressioninneutrophils.49 The that DNA copy number is associated with UTIs and VUR correlation of kidney mRNA expression to DNA copy num- expands genetic variations associated with VUR pathology ber is exciting and usually very elusive in the field of DNA from renal development genes to innate immune genes. In- CNVs. Ideally, we would like to correlate urine protein levels deed, many children with VUR never develop infections and to DNA copy number. Infected urine samples from the seem to have no clinical consequences from the retrograde RIVUR Study were not available. HNP1–3 is not present in flow of urine in the upper urinary tract. Our finding of de- the urine during sterility.40 Ultimately, we would like to be creased DEFA1A3 copy number in children with VUR and able to determine if the source of urinary HNP1–3during UTIs raises the possibility that genetic variations in innate infection is intercalated cells or neutrophils and whether immunity are a risk factor for UTI in the subset of patients levels correlate with DEFA1A3 copy number. Because UTI who experience recurrent infections. is a heterogeneous event with a wide spectrum in degree In the RIVUR Study, 25.4% of children treated with placebo of pyuria, upper tract versus lower tract involvement, had a breakthrough UTI during the 2-year study period. This uroepithelial cellular death and sloughing, and urinary con- rate of UTI was almost twice the rate of children treated with centration and osmolality as well as virulent factors with each antibiotics prophylaxis.29 The patients in the RIVUR Trial ran- uropathogen that may affect neutrophil degranulation or in- domized to placebo and analyzed in this study did not seem to tercalated cell secretion of HNP1–3, designing an appropri- have different breakthrough UTIs on the basis of DEFA1A3 ate experiment would require conditional knockout mouse copy number. Presumably, the anatomic defect of VUR or host models to account for source and then, transgenic mice to characteristics overwhelmed the innate defenses, and there- recapitulate CNVs. Additionally, we aim to determine fore, no differences could be detected. Intriguingly, DEFA1A3 whether DEFA1A3 copy number accounts for differences in copy number is significantly lower in children with VUR on neutrophil bacterial killing between individuals. Finally, antibiotic prophylaxis who develop breakthrough UTIs. quantitative real–time PCR has limitations at discerning Antibiotic prophylaxis seems largely sufficient to prevent re- small copy number differences at high copy numbers, al- current UTIs in children with high DEFA1A3 copy number. though previous studies have shown that PCR-based meth- However, when low DEFA1A3 copy number is present, the odologies are accurate for determining copy numbers in efficacy of antibiotic prophylaxis decreases. Thus, adding an- multiallelic loci.50,51 tibiotic support to individuals with high and low DEFA1A3 copy numbers reveals the added burden caused by reduced a-defensin expression. Therefore, DEFA1A3 copy number de- CONCISE METHODS termination represents a potential strategy to identify patients with VUR who will respond well to antibiotic prophylaxis. Study Design Future prospective studies with appropriately powered ran- Human Subjects Approval domized cohorts are needed to prove this potentially causal Human Subjects Safety Approval. Approval on human subjects was relationship. obtained through Nationwide Children’s Hospital (NCH) Institutional

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Review Board (IRB) Protocols IRB07–00383 and IRB10–00319 and The by the National Cancer Institute.53 No tissue was obtained from Ohio State University Wexner Medical Center IRB Protocol patients with recurrent UTI. Tissue samples were snap frozen or OSUIRB2013H0255. All clinical investigations were conducted accord- preserved as neutral formalin–fixed, paraffin–embedded sections. ing to the principles expressed in the Declaration of Helsinki. Kidney tissues were removed from patients with suspected tumor, and we used adjacent tissue free of disease or inflammation. Genetic Samples Patient Population. Samples were used from the National Institutes DEFA1A3 Copy Typing. of Health–funded RIVUR Study.2,3 Informed consent for genetic Pulse–Field Gel Electrophoresis. Todevelop absolute DEFA1A3 copy samples was obtained as a component of enrollment to the parent number to use for the PCR, PacI-PFGE and Southern blot analysis of RIVUR Study.2,3 The RIVUR Study consisted of 607 children ages DEFA1A3 in nine different human subjects were used. Mononuclear 2–71 months of age presenting with confirmed grades 1–4VURand suspension cells were harvested by centrifugation at 10003g for history of first or second UTI. The patients in the RIVUR Trial were 10 minutes at 4°C and rinsed with PBS. Approximately 2–53106 cells randomized to antimicrobial prophylaxis or placebo and followed were resuspended in 100 ml molten low–gelling temperature agarose for 2 years. UTI guidelines in patients in the RIVUR Trial were adap- to a final concentration of 0.5% (agarose), and the mixture was set ted from the American Academy of Pediatric guidelines and defined in a plug mold. Agarose plugs were then digested with proteinase K as a positive culture with a single organism of $50,000 CFU/ml on a (1.5 mg/ml) to remove cellular proteins in the presence of 10 mM catheter specimen or .100,000 CFU/ml on a clean catch with evi- Tris, 0.5 M EDTA, and 1.0% lauroyl sarcosine (pH 9.5) at 50°C over- dence for pyuria on the urinalysis. DNA from 455 of these children night. After extensive rinses with buffer of 10 mM Tris and 1 mM was obtained by the RIVUR Study. The patients in the RIVUR Trial EDTA, agarose plugs with intact genomic DNA were equilibrated were subgrouped by parameters followed in the RIVUR Study, in- with restriction enzyme buffer, digested with PacIenzyme(15U cluding randomization to placebo versus antibiotic prophylaxis, per reaction) for 2 hours, and resolved with pulsed–field gel electro- fi breakthrough UTIs de ned as UTIs during the study period and phoresis for DNA fragments with sizes between 50 and 400 kb. The fi not including the study eligibility entry UTI, BBD as de ned by agarose gel with resolved genomic DNA fragments was subjected to dysfunctional voiding questionnaire score, and VUR grade along modified Southern blot procedures as described previously.54 fi – with acquired renal scarring de ned by new DMSA positive scars Genomic DNA transferred to nylon membrane was hybridized during the study period. with a DEFA1A3–specific genomic probe labeled by a32P-dCTP Control Population. Control patients were enrolled from patients and subjected to x-ray film autoradiography at 280°C for 3–14 presenting to the NCH Emergency Department. Patients were screened days. Because each segment of duplication for the DEFA1A3 gene is by a study staff interview for history of UTIs or known congenital 19.1 kb in size, the absolute copy number could be determined by the anomalies of the kidney or urinary tract, including VUR, and eligible for size of band on the l-ladder and the number of haplotype bands inclusion if the aforementioned conditions were not present. Written (Figure 6). informed consent was obtained. Because different races and ethnicities High–Throughput Copy Typing. We used multiplex real–time PCR fi can have different copy number pro les, this analysis was limited to using Taqman Dye Chemistry (Applied Biosystems, Foster City, CA) whites.52 Additionally, sex is a confounder in UTI risk, and therefore, and the comparative cycle threshold difference (DDCT)methodto the analysis was limited to girls separate from boys. copy type each individual’stotalDEFA1A3 copy number. The ZNF80 gene served as a two copies per diploid genome reference gene. In- Human Biospecimens. dividuals with known copy number served as calibrators to deter- DNA. Control DNA was obtained from patients presenting to the mine copy number for unknown samples. Calibrators with eight total NCH Emergency Department with no history of UTI. Saliva samples copies were determined by pulsed–field gel electrophoresis. The were obtained using Oragene DNA Collection Kits (DNA Genotek, DEFA1A3 probe was FAM labeled, and the reference ZNF80 probe Kanata, ON, Canada). Saliva samples were transferred to 15-ml was VIC labeled. Briefly, each multiplex reaction consisted of 0.9 mM conical tubes and incubated at 50°C for a minimum of 2 hours. DNA each forward and reverse primers, 100 nM target (FAM-labeled was extracted using prepITzL2P Reagent (PT-L2P-45; Genotek) ac- DEFA1A3) and reference (VIC-labeled ZNF80) probes, 30 ng patient cording to the manufacturer’s manual purification protocol genomic DNA, and 23 Taqman Universal PCR Master Mix. The final for whole samples. After purification and precipitation with etha- volume was adjusted to 20 ml with molecular-grade water. Each nol, DNA was rehydrated with 100 ml Tris-EDTA solution. DNA sample was performed in duplicate. Real-time PCR was performed concentration (nanograms per microliter) and DNA purity using the ABI 7500 Real-Time PCR System, with cycles of 95°C for (a260-to-a280 and a260-to-a230 ratios) were determined using 10 minutes followed by 40 cycles at 95°C for 15 seconds and 60°C fi the Nanodrop 2000 Spectrophotometer (Thermo Fisher Scienti c, for 1 minute. Waltham, MA). Relative quantitation with the DDCTmethod was used to calculate Tissue. We obtained noninfected bladder tissue (n=4) from children the copy number of the DEFA1A3 gene. This method measured the undergoing ureteral reimplantation for reasons other than recur- cycle threshold difference (DCT)betweenDEFA1A3 as a target and rent UTI, such as UPV obstruction. Kidney tissue (n=43) was pro- ZNF80 as a reference and then, compared the DCT values of samples vided by the Cooperative Human Tissue Network, which is funded with the calibrator sample known to have eight copies of the target

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Tissue DEFA1A3 mRNA Expression mRNA expression was analyzed as previously described.30 Briefly, RNA extracted from kidney, bladder, and ureter was reversed transcribed into cDNA using the Verso cDNA Kit (Thermo Scientific). Quantitative real–time PCR was performed using cDNA as thetemplatewithaspecific primer pair (forward primer: 59-CCCTCGCCATCCTTGCTGCC-39 and reverse primer: 59-CTTGAGCCTGGATGCTTTGGAGCC-39). The DEFA1 cDNA clone (cDNA clone MGC: 120826 IMAGE: 7939636; American Type Culture Collection, Manassas, VA) was used as a standard to quantify the mRNA copy numbers for each sample. The PCR conditions were as follows: initial denaturation at 95°C for 10 minutes followed by 40 cycles, with each cycle consisting of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. The reactions were performed using the 7500 Real-Time PCR System (Applied Biosystems). Primers and probes are listed in Table 3.

DEFA1A3 In Situ Hybridization RNA was interrogated in 4-mm paraffin–embedded human kidney sections using the QuantiGene ViewRNA ISH Tissue Assay according to the manufacturer’s protocol (Affymetrix, Santa Clara, CA). Prehy- bridization conditions were optimized with 5 minutes of incubation Figure 6. DEFA1A3 copy number determined by pulse–field in pretreatment solution (90°C) and 5 minutes of treatment with electrophoresis. Each segment of duplication for a DEFA1A3 protease QF (40°C). Sections were hybridized for 3 hours (40°C) gene is 19.1 kb in size. Different combinations of haplotype copy with a viewRNA Probe Set designed against the DEFA1A3 mRNA. numbers are shown. Subjects with single fragments are homo- The probes were then amplified (PreAmplifier and Amplifier mole- zygous; subjects with two fragments are heterozygous. CN, copy cules), conjugated to alkaline phosphatase (Label Probe 1-AP), and number; H1, haplotype 1; H2, haplotype 2; HCN, haplotype copy bound with Fast Red Substrate. Finally, sections were counterstained number; total GCN, total gene copy number of DEFA1A3 in a with hematoxylin and imaged with the EVOS FL Auto Cell Imaging diploid genome. System (Life Technologies, Grand Island, NY). sequence. Known samples with DEFA1A3 copy numbers of six, seven, fl and 11 were included on each plate as quality controls: Immuno uorescence After deparaffinizion, rehydration and permeabilization with 0.1%

DCT ¼ CTðtargetÞ 2 CTðreferenceÞ; Triton X-100, antigen with Tris-EDTA (pH 9.0), and blocking superblock solution (Scy Tek, Logan, UT) were performed. Sections DD ¼ D ð Þ 2 D ð Þ; CT CT sample CT calibrator and were incubated with anti-human HNP1–3 (1:300 dilution; Hycult total DEFA1A3 copy numberðsampleÞ¼copy numberðcalibratorÞ Biotech, Plymouth Meeting, PA) overnight at 4°C. After washing – ðDD Þ with PBS, 10% superblock sections were incubated with anti mouse 32 CT : IgG-AF488 (1:200 dilution; Jackson ImmunoResearch Laboratories,

Copy numbers were rounded up or down to the closest integer. Table 3. Genotyping primers Nucleic Acid and Protein Isolation from Human Kidney Primer Name Primer Sequences Samples DEFA1 59-CCTGTCCCAGGCTCAAGGA-39 Kidney tissue samples were divided into 100-mg pieces. Tissue forward samples were homogenized in a bullet blender (BBY-24M; Next DEFA1 59-CATAGCGACGTTCTCCTGCAAT-39 Direct,AverillPark,NY)usingtwoor three3.2-mmstainlesssteelbeads reverse and five or six 2.9-mm-diameter zirconium oxide beads (Next Direct) Probe FAM-GCTATTGCAGAATACC-MGB ZNF80 59-TCTGTCTTCATCCAGCATCG-39 under standard conditions. The extracted DNA was resuspended in forward buffer 10 mM Tris-HCl and 1 mM EDTA (pH. 8.0). ZNF80 59-CTTTGCACTCGTAGGGCTTT-39 RNAwas extracted using the Trizol method. Adjacent pieces of the reverse same kidney specimens used for DNA and RNA extraction were Probe VIC-GAAGACCTTCACCTACCGCTCTGTTTT-MGB homogenized in RIPA buffer with protease inhibitors to achieve tissue FAM, 6-carboxyfluorescein; MGB, minor groove binder; VIC, 4,7,29-trichloro- lysate for protein analysis. 79-phenyl-6-carboxyfluorescein.

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West Grove, PA) for 30 minutes at room temperature. For Aquaporin the Children’s Hospital of Philadelphia Clinical and Translational staining, sections were labeled for 30 minutes at room temperature Science Award (UL1TR000003) both from the National Center for with a 1:400 dilution of anti–AQP2-Rhodamine antibody (anti– Research Resources, now at the National Center for Advancing AQP2-ATTO-550; Alomone Laboratory, Jerusalem, Israel) and coun- Translational Sciences, National Institutes of Health. We also thank the terstained with DAPI (H-1500; Vector Laboratories, Burlingame, CA) Cooperative Human Tissue Network for tissues used in this study. visualized on a Keyence Microscope (Keyence, Osaka, Japan) with We thank the Nationwide Children’s Hospital Emergency Depart- appropriate ﬁlters and brightness along with contrast adjusted with ment Research Team and Ariel Cohen for meticulous collection Keyence imaging software. Secondary antibody alone was used as a of control samples and DNA extraction. Finally, we thank John negative control. Armour for his thoughtful insight on characterizing this challeng- ing gene locus. Replicates and Outliers A.L.S., P.D.B., K.M.M., G.C.T., N.S., J.R.M., and D.S.H. were all Genotyping experiments were performed in triplicate; otherwise, all funded by National Institutes of Health (NIH) grant 1RC4DK090937-01. otherexperiments were performed in duplicate. No outlying datawere J.D.S. is funded by NIH grant K08 DK094970-01. excluded.

Statistical Analyses DISCLOSURES Gene copy number analysis was done using t tests to assess the dis- None. tribution difference (mean values) of copy number between independent two–sample groups (e.g., patients in the RIVUR Trial versus controls, entry versus recurrence groups, E. coli versus non-E. coli, REFERENCES and breakthrough UTI risk between the antibiotic and the placebo groups). To perform comparisons between ratios of groups (e.g.,pa- 1. Williams G, Fletcher JT, Alexander SI, Craig JC: Vesicoureteral reflux. – tients in the RIVUR Trial versus controls and breakthrough UTI risk J Am Soc Nephrol 19: 847 862, 2008 2. Carpenter MA, Hoberman A, Mattoo TK, Mathews R, Keren R, Chesney between the antibiotic and the placebo groups) with low, medium, or RW, Moxey-Mims M, Greenfield SP; RIVUR Trial Investigators: The high DEFA1A3 copy number, the chi-squared test was performed, RIVUR trial: Profile and baseline clinical associations of children with provided that $80% of the cells have an expected frequency of greater vesicoureteral reflux. Pediatrics 132: e34–e45, 2013 than or equal to five and no cell has an expected frequency ,1.0; 3. Hoberman A, Greenfield SP, Mattoo TK, Keren R, Mathews R, Pohl HG, otherwise, the Fisher exact test was used. To perform statistical anal- Kropp BP, Skoog SJ, Nelson CP, Moxey-Mims M, Chesney RW, Carpenter MA; RIVUR Trial Investigators: Antimicrobial prophylaxis for ysis for HNP1–3/DEFA1A3, expression differences were evaluated children with vesicoureteral reflux. N Engl J Med 370: 2367–2376, – with the Mann Whitney test. Linear regression analysis was per- 2014 formed to reveal the linear relationship between CNV and mRNA, 4. Keren R, Shaikh N, Pohl H, Gravens-Mueller L, Ivanova A, Zaoutis L, and confidence intervals are presented. Toassess the risk for recurrent Patel M, deBerardinis R, Parker A, Bhatnagar S, Haralam MA, Pope M, UTI related to CNVs, the group receiving antibiotics, and their in- Kearney D, Sprague B, Barrera R, Viteri B, Egigueron M, Shah N, Hoberman A: Risk factors for recurrent urinary tract infection and renal teraction, we performed logistical regression analysis with an adjust- scarring. Pediatrics 136: e13–e21, 2015 ment for VUR grade and BBD using the function glm in R. Statistical 5. Fidan K, Kandur Y, Buyukkaragoz B, Akdemir UO, Soylemezoglu O: analyses were done using stats in the open source statistical software R Hypertension in pediatric patients with renal scarring in association with (www.r-project.org), Vassarstats (http://vassarstats.net), and Prism vesicoureteral reflux. Urology 81: 173–177, 2013 software (GraphPad Software, La Jolla, CA). All statistical tests were 6. Seikaly MG, Ho PL, Emmett L, Fine RN, Tejani A: Chronic renal fi two sided, with a significant level of 95% (i.e., P value ,0.05). insuf ciency in children: The 2001 Annual Report of the NAPRTCS. Pediatr Nephrol 18: 796–804, 2003 7. Quirino IG, Diniz JS, Bouzada MC, Pereira AK, Lopes TJ, Paixão GM, Barros NN, Figueiredo LC, Cabral AC, Simões e Silva AC, Oliveira EA: Clinical course of 822 children with prenatally detected nephrouropathies. ACKNOWLEDGMENTS Clin J Am Soc Nephrol 7: 444–451, 2012 8. Wang HH, Gbadegesin RA, Foreman JW, Nagaraj SK, Wigfall DR, This report is dedicated to the memory of Russell W. Chesney for his Wiener JS, Routh JC: Efficacy of antibiotic prophylaxis in children with enthusiastic support and sage advice for this study. We thank the vesicoureteral reflux: Systematic review and meta-analysis. J Urol 193: – Randomized Intervention for Children with Vesicoureteral Reflux 963 969, 2015 9. Weichhart T, Haidinger M, Hörl WH, Säemann MD: Current concepts of Trial Investigators and the National Institute of Diabetes and Digestive molecular defence mechanisms operative during urinary tract infection. and Kidney Diseases for their support of this project. This researchwas Eur J Clin Invest 38[Suppl 2]: 29–38, 2008 supported by grants U01 DK074059, U01 DK074053, U01 DK074082, 10. Zasloff M: Antimicrobial peptides, innate immunity, and the normally U01 DK074064, U01 DK074062, U01 DK074063 from the National sterile urinary tract. JAmSocNephrol18: 2810–2816, 2007 Institute of Diabetes and Digestive and Kidney Diseases, National 11. Spencer JD, Schwaderer AL, Becknell B, Watson J, Hains DS: The innate immune response during urinary tract infection and pyelonephri- Institutes of Health, Department of Health and Human Services. This tis. Pediatr Nephrol 29: 1139–1149, 2014 trial was also supported by the University of Pittsburgh Clinical and 12. Ali AS, Townes CL, Hall J, Pickard RS: Maintaining a sterile urinary tract: Translational Science Award(UL1RR024153 and UL1TR000005), and The role of antimicrobial peptides. JUrol182: 21–28, 2009

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