The orchestration of gene expression and the editing role of microRNA.

In this short educational communication the ESPU Research Committee presents the role of non-coding RNA and how these can affect gene expression. In particular we discuss the role of microRNA on post transcriptional changes and how these may cause pathological conditions within Pediatric Urology and how microRNA could be useful in future clinical practice.

Measurement and initial characterization of leukocyte telomere length in 474,074 participants in UK Biobank.

Leukocyte telomere length (LTL) is a proposed marker of biological age. Here we report the measurement and initial characterization of LTL in 474,074 participants in UK Biobank. We confirm that older age and male sex associate with shorter LTL, with women on average ~7 years younger in 'biological age' than men. Compared to white Europeans, LTL is markedly longer in African and Chinese ancestries. Older paternal age at birth is associated with longer individual LTL. Higher white cell count is associated with shorter LTL, but proportions of white cell subtypes show weaker associations. Age, ethnicity, sex and white cell count explain ~5.5% of LTL variance. Using paired samples from 1,351 participants taken ~5 years apart, we estimate the within-individual variability in LTL and provide a correction factor for this. This resource provides opportunities to investigate determinants and biomedical consequences of variation in LTL.

The association of postnatal urinary tract dilation risk score with clinical outcomes.

BACKGROUND: There are limited data on the predictive value of the consensus urinary tract dilation (UTD) score with respect to subsequent clinical diagnoses. We sought to define the relationship between postnatal UTD risk score and clinical outcomes during childhood. METHODS: Complete ultrasound image sets from a random selection of infants aged 0-90 days undergoing initial ultrasound at a single institution for prenatal hydronephrosis between 2012 and 2014 were assigned a UTD score by 1 pediatric urologist and 1 pediatric radiologist. Urinary tract dilation risk score was analyzed for association with a composite outcome comprising urinary tract infection, vesicoureteral reflux (VUR), ureteropelvic junction obstruction, non-refluxing megaureter (NRM), ureterocele, bladder outlet obstruction (BOO), and chronic kidney disease. Surgical intervention and resolution of UTD were evaluated separately. Descriptive and survival analyses were performed. RESULTS: Urinary tract dilation scores for 494 subjects were P0 in 23.5%, P1 in 26.5%, P2 in 23.5%, and P3 in 26.5%. Seventy-four percent were male. Median age at initial imaging was 28 days; median follow-up was 19.8 months. The composite outcome occurred in 138 of 494 patients (27.9%) and varied significantly (p < 0.001) by UTD score: 11.2% for P0, 10.7% for P1, 29.3% for P2, and 58.8% for P3. On survival analysis (Summary Figure), higher UTD grade was significantly associated with the composite outcome (hazard ratio for P3 vs. P0 was 7.4 [95% CI: 3.44-15.92, p < 0.001]). Urinary tract infection and VUR diagnosis varied by UTD score (p = 0.03 and p < 0.001, respectively). Ureteropelvic junction obstruction was diagnosed (based on MAG3 results) in 6.3% of patients, 84% of whom were P3. Non-refluxing megaureter was diagnosed in 7.7%. Ureterocele and BOO were uncommon (1.4%, and 0.6%, respectively). Surgical intervention was also associated with UTD risk, with 46% of P3 undergoing surgery vs. 1% of P0, 1% of P1, and 6% of P2 (p < 0.001). Resolution of UTD occurred in 41% (median 10.1 months) and varied significantly by UTD risk (p < 0.001). DISCUSSION: Urinary tract dilation risk score is associated with clinical events, although ascertainment bias may influence some of the differences in outcomes, particularly for VUR, because VCUG utilization varied by the UTD group. The lack of any significant difference in outcomes between patients with UTD P0 versus P1 suggests that the P1 category could be eliminated as it does not meaningfully distinguish between outcome risk. CONCLUSIONS: Higher UTD risk scores are strongly associated with genitourinary diagnoses during the first two years of life.

The association between continuous antibiotic prophylaxis and UTI from birth until initial postnatal imaging evaluation among newborns with antenatal hydronephrosis.

INTRODUCTION: There is a lack of consensus regarding the use of continuous antibiotic prophylaxis (CAP) during the interval between birth and initial postnatal imaging in infants with a history of antenatal urinary tract dilation (AUTD). OBJECTIVE: To determine the incidence of urinary tract infection (UTI), and the association between CAP use and UTI during the interval between birth and the first postnatal renal ultrasound (RUS) in infants with AUTD. STUDY DESIGN: A single-institution, retrospective cohort study of newborns with a history of AUTD. Infants undergoing RUS within 3 months of birth for an indication of 'hydronephrosis' between 2012 and 2014 were identified. A random sample of 500 infants was selected; six were excluded for concomitant congenital anomalies. Baseline patient (sex, race, insurance) and clinical characteristics (circumcision status, UTD risk score, receipt of CAP, UTI prior to RUS, age at UTI, and age at RUS) were collected via retrospective chart review. Descriptive statistics were calculated. To adjust for receipt of CAP, propensity score adjusted univariate logistic regression for UTI based on CAP status was performed. RESULTS: Among the 494 infants with AUTD, 157 (32%) received CAP. Infants with normal/low-risk UTD scores were less likely to receive CAP than those with medium/high-risk UTD (23% vs 77%; P < 0.001). There was no difference in CAP based on sex, insurance, or circumcision status (among 260/365 males with known circumcision status). Overall, seven infants (1.4%) developed UTI prior to imaging: six (1.8%) without CAP vs one (0.64%) with CAP (P = 0.44). The median age at UTI was 59 days (range 2-84); among those with UTI, initial imaging occurred significantly later (66 vs 28 days; P = 0.001). The propensity score adjusted odds of developing UTI with CAP (vs without) was 0.93 (95% CI 0.10-8.32; P = 0.95). The Summary Figure describes the infants with UTI. CONCLUSION: The incidence of UTI prior to initial neonatal imaging in newborns with AUTD was low. Use of CAP was not associated with UTI incidence after adjusting for UTD severity. Routine use of CAP in newborns with AUTD prior to initial imaging may be of limited benefit in most patients.

Urology mythbusters: Radiation and radiophobia.

In this episode of Mythbusters we critically examine the premise that there is strong biological and epidemiologic evidence that radiation exposure at levels associated with modern genitourinary diagnostic imaging increases the risk of subsequent malignancy, especially in children.

Determinants of day-night difference in blood pressure, a comparison with determinants of daytime and night-time blood pressure.

Blunted day-night difference in blood pressure (BP) is an independent cardiovascular risk factor, although there is limited information on determinants of diurnal variation in BP. We investigated determinants of day-night difference in systolic (SBP) and diastolic (DBP) BP and how these compared with determinants of daytime and night-time SBP and DBP. We analysed the association of mean daytime, mean night-time and mean day-night difference (defined as (mean daytime-mean night-time)/mean daytime) in SBP and DBP with clinical, lifestyle and biochemical parameters from 1562 adult individuals (mean age 38.6) from 509 nuclear families recruited in the GRAPHIC Study. We estimated the heritability of the various BP phenotypes. In multivariate analysis, there were significant associations of age, sex, markers of adiposity (body mass index and waist-hip ratio), plasma lipids (total and low-density lipoprotein cholesterol and triglycerides), serum uric acid, alcohol intake and current smoking status on daytime or night-time SBP and/or DBP. Of these, only age (P=4.7 × 10-5), total cholesterol (P=0.002), plasma triglycerides (P=0.006) and current smoking (P=3.8 × 10-9) associated with day-night difference in SBP, and age (P=0.001), plasma triglyceride (P=2.2 × 10-5) and current smoking (3.8 × 10-4) associated with day-night difference in DBP. 24-h, daytime and night-time SBP and DBP showed substantial heritability (ranging from 18-43%). In contrast day-night difference in SBP showed a lower heritability (13%) while heritability of day-night difference in DBP was not significant. These data suggest that specific clinical, lifestyle and biochemical factors contribute to inter-individual variation in daytime, night-time and day-night differences in SBP and DBP. Variation in day-night differences in BP is largely non-genetic.

Evaluation of the contralateral inguinal ring in clinically unilateral inguinal hernia: a systematic review and meta-analysis.

PURPOSE: The management of the contralateral inguinal canal in children with clinical unilateral inguinal hernia is controversial. Our objective was to systematically review the literature regarding management of the contralateral inguinal canal. METHODS: We searched MEDLINE, EMBASE, and Cochrane databases (1940-2011) using 'hernia' and 'inguinal' and either 'pediatric,' 'infant,' or 'child,' to identify studies of pediatric (age ≤21 years) patients with inguinal hernia. Among clinical unilateral hernia patients, we assessed the number of cases with contralateral patent processus (CPP) and incidence of subsequent clinical metachronous contralateral hernia (MCH). We evaluated three strategies for contralateral management: expectant management, laparoscopic evaluation or pre-operative ultrasound. Pooled estimates of MCH or CPP were generated with random effects by study when heterogeneity was found (I(2) > 50 %, or Cochrane's Q p ≥ 0.10). RESULTS: We identified 2,477 non-duplicated studies, 129 of which met our inclusion criteria and had sufficient information for quantitative analysis. The pooled incidence of MCH after open unilateral repair was 7.3 % (95 % CI 6.5-8.1 %). Laparoscopic examination identified CPP in 30 % (95 % CI 26-34 %). Lower age was associated with higher incidence of CPP (p < 0.01). The incidence of MCH after a negative laparoscopic evaluation was 0.9 % (95 % CI 0.5-1.3 %). Significant heterogeneity was found in studies and pooled estimates should be interpreted with caution. CONCLUSIONS: The literature suggests that laparoscopically identified CPP is a poor indicator of future contralateral hernia. Almost a third of patients will have a CPP, while less than one in 10 will develop MCH when managed expectantly. Performing contralateral hernia repair in patients with CPP results in overtreatment in roughly 2 out of 3 patients.

Should relative survival be used with lung cancer data?

BACKGROUND: Under certain assumptions, relative survival is a measure of net survival based on estimating the excess mortality in a study population when compared with the general population. Background mortality estimates are usually taken from national life tables that are broken down by age, sex and calendar year. A fundamental assumption of relative survival methods is that if a patient did not have the disease of interest then their probability of survival would be comparable to that of the general population. It is argued, as most lung cancer patients are smokers and therefore carry a higher risk of smoking-related mortalities, that they are not comparable to a population where the majority are likely to be non-smokers. METHODS: We use data from the Finnish Cancer Registry to assess the impact that the non-comparability assumption has on the estimates of relative survival through the use of a sensitivity analysis. RESULTS: Under realistic estimates of increased all-cause mortality for smokers compared with non-smokers, the bias in the estimates of relative survival caused by the non-comparability assumption is negligible. CONCLUSION: Although the assumption of comparability underlying the relative survival method may not be reasonable, it does not have a concerning impact on the estimates of relative survival, as most lung cancer patients die within the first 2 years following diagnosis. This should serve to reassure critics of the use of relative survival when applied to lung cancer data.

Estimating the crude probability of death due to cancer and other causes using relative survival models.

Relative survival is used extensively in population-based cancer studies to measure patient survival correcting for causes of death not related to the disease of interest. An advantage of relative survival is that it provides a measure of mortality associated with a particular disease, without the need for information on cause of death. Relative survival provides a measure of net mortality, i.e. the probability of death due to cancer in the absence of other causes. This is a useful measure, but it is also of interest to measure crude mortality, i.e. the probability of death due to cancer in the presence of other causes. A previous approach to estimate the crude probability of death in population-based cancer studies used life table methods, but we show how the estimates can be obtained after fitting a relative survival model. We adopt flexible parametric models for relative survival, which use restricted cubic splines for the baseline cumulative excess hazard and for any time-dependent effects. We illustrate the approach using an example of men diagnosed with prostate cancer in England and Wales showing the differences in net and crude survival for different ages.

Should Asian men be included in abdominal aortic aneurysm screening programmes?

INTRODUCTION: A national AAA screening programme for men aged 65 is shortly to be implemented in England. Trials that have provided evidence for this screening programme have not included information on ethnicity. Hence their findings may not be applicable to ethnically diverse populations. REPORT: This study retrospectively looked at the prevalence of AAA in men aged 65, from different ethnic backgrounds in our city's current screening programme.19014 men (Caucasians n=18,431, Asian n=446, others n=137) were screened. Prevalence was 4.69% (4.39-5% 95% CI), and 0.45% (0.054-1.161% 95% CI) in Caucasians and Asians respectively (Fisher's exact test: P<0.0001). DISCUSSION: Prevalence of AAAs in men aged 65 of Asian origin appears to be low and so increases uncertainty about cost-effectiveness of screening Asian men.

Modern staged repair of bladder exstrophy: a contemporary series.

OBJECTIVE: Many changes have occurred in the treatment of bladder exstrophy over the last few years and many repairs are now offered. The purpose of this study was to evaluate long-term outcomes in a select group of patients in whom modern staged repair (MSRE) was undertaken. PATIENTS AND METHODS: From an institutionally approved database were extracted 189 patients who had undergone primary closure between 1988 and 2004. The records of 131 patients (95 males) who underwent MSRE with a modified Cantwell-Ransley repair by a single surgeon in 1988-2004 were reviewed with a minimum 5-year follow up. RESULTS: Sixty-seven patients with a mean age of 2 months (range 6 h to 4 months) underwent primary closure, and 18 underwent osteotomy at the same time. Mean age at epispadias repair was 18 months (8-24). Mean age at bladder neck reconstruction (BNR) was 4.8 years (40-60 months) with a mean capacity of 98 cc (75-185). Analysis of bladder capacity prior to BNR revealed that patients with a mean capacity greater than 85 cc median had better outcomes. Seventy percent (n=47) are continent day and night and voiding per urethra without augmentation or intermittent catheterization. Social continence defined as dry for more than 3h during the day was found in 10% (n=7). Six patients required continent diversion after failed BNR. Seven patients are completely incontinent. The mean time to daytime continence was 14 months (4-23) and the mean time to night-time continence was 23 months (11-34). No correlation was found between age at BNR and continence. CONCLUSIONS: Patients with a good bladder template who develop sufficient bladder capacity after successful primary closure and epispadias repair can achieve acceptable continence without bladder augmentation and intermittent catheterization.

EUTUBS: a mnemonic for the complete endoscopic examination of the lower urinary tract.

Urethrocystoscopy is now routinely done in standard Urological practice. The availability of the flexible cystoscope for outpatient procedures has further increased the number of cystoscopists. However, there are currently no formal training schedules for urethrocystoscopy. This mnemonic has been developed to serve as a template for complete endoscopic examination of the lower urinary tract to which the cystoscopist may refer when undertaking this operation. It focuses on the common abnormalities that the endoscopist may encounter, and is not intended to be an exhaustive list of all abnormalities of the lower urinary tract. Furthermore, it is not meant to obviate the need for practical training of those wishing to carry out the procedure as part of their clinical practice.

Sales and science: changing patterns of pharmaceutical and medical device advertising in peer reviewed urology publications, 1975-2000.

PURPOSE: The effects of advertising on urological practice are controversial. We studied patterns of pharmaceutical and medical device marketing in peer reviewed urological journals in 1975 and 2000. MATERIALS AND METHODS: Pharmaceutical and medical device advertising in 1 European and 2 American peer reviewed urological journals were evaluated in 4 randomly selected issues of each journal published in 1975 and 2000, respectively. Advertising quantity and the qualitative characteristics of each advertisement were analyzed. RESULTS: We analyzed 574 advertisements in 24 issues. Advertising decreased between 1975 and 2000 based on the number of pages per issue (55.3 to 31.9, p = 0.04), number of advertisements per issue (30.4 to 17.4, p = 0.0098) and the ratio of advertising-to-scientific pages (0.399 to 0.151, p = 0.0016). Mean advertisement length was stable at 1.8 pages. The top 3 advertisers in 1975 were Eaton, Roche and Warner compared with Pfizer, AstraZeneca and Merck in 2000. Advertising for antibiotics comprised 70.3% of all pharmaceutical advertisements in 1975 but only 15.2% in 2000 (p = 0.0001), while advertising for benign prostatic hyperplasia, erectile dysfunction and hormonal therapy increased sharply. Nutritional supplement marketing increased from 0.5% of all advertisements in 1975 to 4.3% in 2000 (p = 0.0026). The incidence of advertisements citing peer reviewed literature increased from 16.7% to 33% (p = 0.0001) with a greater increase in the European than in the American journals. CONCLUSIONS: Advertising in peer reviewed urological journals has decreased since 1975 and fewer companies now market more products. Few advertisements cite the scientific literature. Better understanding of pharmaceutical marketing patterns may improve awareness of these efforts to influence physician practice.

GSTA1 expression in normal, preneoplastic, and neoplastic human prostate tissue.

BACKGROUND: Glutathione S-transferases (GSTs), inducible enzymes that catalyze the detoxification of reactive electrophiles and oxidants, protect against neoplastic transformation. Prostatic adenocarcinoma and high-grade prostatic intraepithelial neoplasia (HGPIN) fail to express GSTP1, a major class of GST. This failure of expression is associated with methlyation of the GSTP1 promoter, a somatic alteration proposed to be a critical step in prostatic carcinogenesis. However, simple atrophy and post-atrophic hyperplasia-proliferative lesions associated with chronic inflammation, which we have termed "proliferative inflammatory atrophy" (PIA)-express elevated levels of GSTP1. We postulated that this increase in GSTP1 expression in PIA occurs in response to increased oxidative stress. We examined the expression of another major class of GST, GSTA1, in the human prostate. METHODS: We performed immunohistochemistry against GSTA1 on formalin-fixed radical prostatectomies (n = 45). A stereological grid point counting method was used to estimate the percent of cells staining positive for GSTA1 in normal prostate, PIA, HGPIN, and adenocarcinoma. RESULTS: In contrast to GSTP1, normal peripheral zone epithelium was virtually devoid of GSTA1. Strikingly, though, epithelial cells in PIA demonstrated strong staining for GSTA1 (median of percent of cells staining positive = 44) as compared to those in normal peripheral zone (median = 3.0, P <.00001), HGPIN (median = 2.9, P <.00001), and adenocarcinoma (median = 3.8, P <.00001). Variations in GSTA1 were also detected between normal anatomic zones: the central zone showed an increase in the percentage of cells staining positive (median = 20.9) as compared to the transition (median = 0.47, P <.0002) and the peripheral (P <.0001) zones. CONCLUSIONS: Expression of GSTA1 is increased in PIA, supporting the concept that cells within these lesions are subject to localized increases in oxidative stress. Low levels of GSTA1 and GSTP1 in HGPIN and adenocarcinoma suggest a broad lack of detoxification activity in these cells, which may be associated with carcinogenesis in the prostate.

Comparison of shockwave lithotripsy outcomes in patients receiving sufentanil or lidocaine spinal anesthesia.

PURPOSE: To determine whether the use of intrathecal sufentanil, which allows the patient to move during shockwave lithostripsy (SWL), affects treatment outcomes and operative and recovery times compared with standard lidocaine spinal anesthesia. PATIENTS AND METHODS: We retrospectively studied a series of 62 SWL procedures performed on an unmodified Dornier HM3 lithotripter. The mean calculus size was 10.7 mm. There were 46 renal calculi, 13 ureteral calculi, and 4 patients with calculi in both locations. Of the 63 procedures, 25 were performed using intrathecal sufentanil alone, and 37 were performed with intrathecal lidocaine with or without additional agents. We compared treatment outcomes, as well as treatment time, fluoroscopy time, postanesthesia care unit (PACU) time, time to voiding, and time to ambulation. RESULTS: Sufentanil use was associated with a significantly higher rate of successful treatment, defined as residual fragments absent or <4 mm on follow-up imaging, compared with lodocaine: 68% v. 40% (p = 0.0394). There was no significant difference between the groups in treatment time or fluoroscopy time. Use of sufentanil was associated with significantly shorter PACU time, time to ambulation, and time to voiding postoperatively. These differences persisted when men and women were analyzed separately, although the differences were less significant in women. CONCLUSIONS: The use of intrathecal sufentanil for anesthesia during SWL does not adversely affect treatment outcome; it is, in fact, associated with better outcomes. The advantages of this agent in shortening recovery times and in easing patient transfer into the HM3 gantry argue for increasing its use.

Protection against 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine cytotoxicity and DNA adduct formation in human prostate by glutathione S-transferase P1.

The prostate has been identified as a target for 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced carcinogenesis. Humans are exposed to PhIP through ingestion of well-done cooked meats, and there is evidence from epidemiological studies that implicates red meat consumption in prostate carcinogenesis. The alpha and pi class isoforms of glutathione S-transferases (GSTs) have been shown to inhibit adduction of activated PhIP metabolites to DNA in cell-free systems. In humans, silencing of GST pi(GSTP1) through CpG island hypermethylation is found in nearly all prostate carcinomas and is believed to be an early event in prostate carcinogenesis. We hypothesized that suppressed GSTP1 expression in prostate cells would increase their vulnerability to cytotoxicity and DNA adduct formation mediated by activated PhIP metabolites. To test this hypothesis, the human prostate adenocarcinoma cell line, LNCaP, which contains a silenced GSTP1 gene, was genetically modified to constitutively express high levels of GSTP1. Both LNCaP and LNCaP-GSTP1 cells exposed to N-OH-PhIP, but not parent PhIP, for 24 h showed a dose-dependent decrease in cell viability. GSTP1-overexpressing cells had LC50s 30-40% higher than cells transfected with the vector alone. PhIP-DNA adducts isolated from LNCaP-derived cells and primary human prostate tissue cultures exposed to N-OH-PhIP were analyzed by liquid chromatography/electrospray ionization mass spectrometry. Primary cultures of human prostate tissue and LNCaP-GSTP1 cells had approximately 50% lower adduct levels than parental LNCaP and vector control cells. Bioactivation assays using LNCaP cytosols showed that enzymatic activation of N-OH-PhIP to a DNA binding species was dependent on ATP and could be inhibited by recombinant human GSTP1 in the presence of glutathione. This evidence confirms that N-OH-PhIP can be bioactivated to a DNA binding species in human prostate and human prostate-derived cells. These observations provide the basis for using LNCaP and LNCaP-GSTP1 cells as a model system for studying the role of this enzyme in protection against N-OH-PhIP induced DNA damage in prostate carcinogenesis. Loss of GSTP1 expression in human prostate may, therefore, enhance its susceptibility to carcinogenic insult by compounds such as N-OH-PhIP. Conversely, induction of GSTs in early-stage prostate carcinogenesis may be a useful protective strategy.

Preneoplastic prostate lesions: an opportunity for prostate cancer prevention.

Environmental factors, especially the diet, play a prominent role in the epidemic of prostate cancer (PCA), in the United States. Many candidate dietary components have been proposed to influence human prostatic carcinogenesis, including fat, calories, fruits and vegetables, anti-oxidants, and various micronutrients, but the specific roles dietary agents play in promoting or preventing PCA remain controversial. We have collected evidence to suggest that GSTP1, the gene encoding the pi-class glutathione S-transferase (GST), may serve a "caretaker" function for prostatic cells. Although GSTP1 can be detected in normal prostatic epithelium, in almost all PCA cases, PCA cells fail to express GSTP1 polypeptides, and lack of GSTP1 expression most often appears to be the result of somatic "CpG island" DNA methylation changes. Loss of GSTP1 function also appears to be characteristic of prostatic epithelial neoplasia (PIN) lesions, thought to represent PCA precursors. We have recently learned that a new candidate early PCA precursor lesion, proliferative inflammatory atrophy (PIA), characterized by proliferating prostatic cells juxtaposed to inflammatory cells, contains epithelial cells that express high levels of GSTP1. These findings have formed the basis for a new model of prostatic carcinogenesis, in which prostatic cells in PIA lesions, subjected to a barrage of inflammatory oxidants, induce GSTP1 expression as a defense against oxidative genome damage. When cells with defective GSTP1 genes appear amongst the PIA cells, such cells become vulnerable to oxidants and electrophiles that inflict genome damage that tends to promote neoplastic transformation to PIN and PCA cells. Subsequently, PIN and PCA cells with defective GSTPI genes remain vulnerable to similar stresses tending to promote malignant progression. This new model for prostatic carcinogenesis has implications for the design of new prostate cancer prevention strategies. Rational prevention approaches might include: (i) restoration of GSTPI expression via treatment with inhibitors of CpG methylation, (ii) compensation for inadequate GSTPI activity via treatment with inducers of general GST activity, and (iii) abrogation of genome-damaging stresses via avoidance of exogenous carcinogens and/or reduction of endogenous carcinogenic (particularly oxidant) stresses.

Activation of epithelial Na(+) channel activity in the rabbit urinary bladder by cAMP.

The rabbit urinary bladder actively absorbs Na(+) from the urine. The rate-limiting step in this process is the diffusion of Na(+) across the apical membrane of bladder epithelial cells, mediated by amiloride-sensitive epithelial Na(+) channels. We have investigated the effects of cAMP on epithelial Na(+) channel activity in the rabbit bladder by measuring the amiloride-sensitive short-circuit current across bladders mounted in Ussing chambers. Three agents that raise intracellular cAMP levels (forskolin, dibutyryl-cAMP and 3-isobutyl-1-methylxanthine (IBMX)) increased the amiloride-sensitive short-circuit current relative to control preparations. The forskolin-induced increase in amiloride-sensitive short-circuit current was significantly inhibited by the vesicle fusion inhibitor brefeldin A and the protein synthesis inhibitor cycloheximide. These findings, together with the magnitude and protracted time course of the cAMP effects, suggests that cAMP stimulates the insertion of new Na(+) channels into the apical membrane of the rabbit bladder epithelium.

ELISA of IgG antibody to oxidized low-density lipoprotein: effects of blocking buffer and method of data expression.

We describe an ELISA for serum IgG antibodies against malondialdehyde-modified low-density lipoprotein (mLDL). Optimal antigen concentration, serum dilution, and dilution of enzyme-conjugated second antibody were 25 mg/L, 1:250, and 1:5000, respectively, when 5 g/L human serum albumin was used for blocking. When data were expressed as mLDL/LDL (the ratio of IgG binding to mLDL vs LDL), within-run and between-run CVs were 7.0% and 8.9%, respectively. Antibody concentrations expressed as mLDL/LDL or as mLDL-LDL (the difference between IgG binding to mLDL and to LDL) were higher in women with systemic lupus erythematosus (n = 20) than in controls (n = 20) (P < 0.001). With bovine serum albumin or Superblock blocking buffers, only the mLDL-LDL data were significant. Thus, the choice of blocking agent and the method of data expression should be carefully considered when assaying IgG antibodies against mLDL.