Phase 1/2 AAV5-hRKp.RPGR (Botaretigene Sparoparvovec) Gene Therapy: Safety and Efficacy in RPGR-associated X-linked Retinitis Pigmentosa.

PURPOSE: To assess the safety and efficacy of AAV5-hRKp.RPGR in participants with retinitis pigmentosa GTPase regulator (RPGR)-associated X-linked retinitis pigmentosa (XLRP). DESIGN: Open-label, phase 1/2 dose escalation/expansion study (NCT03252847). METHODS: Males (≥5 years old) with XLRP-RPGR were evaluated. In the dose escalation phase, subretinal AAV5-hRKp.RPGR (low: 1.0×1011 vg/ml; intermediate: 2.0×1011 vg/ml; high: 4.0×1011 vg/ml) was administered to the poorer-seeing eye (n = 10). Dose confirmation (intermediate dose) was carried out in 3 pediatric participants. In the dose expansion phase, 36 participants were randomized 1:1:1 to immediate (low or intermediate dose) or deferred (control) treatment. The primary outcome was safety. Secondary efficacy outcomes included static perimetry, microperimetry, vision-guided mobility, best corrected visual acuity, and contrast sensitivity. Safety and efficacy outcomes were assessed for 52 weeks for immediate treatment participants and 26 weeks for control participants. RESULTS: AAV5-hRKp.RPGR was safe and well tolerated, with no reported dose-limiting events. Most adverse events (AEs) were transient and related to the surgical procedure, resolving without intervention. Two serious AEs were reported with immediate treatment (retinal detachment, uveitis). A third serious AE (increased intraocular pressure) was reported outside the reporting period. All ocular inflammation-related AEs responded to corticosteroids. Treatment with AAV5-hRKp.RPGR resulted in improvements in retinal sensitivity and functional vision compared with the deferred group at Week 26; similar trends were observed at Week 52. CONCLUSIONS: AAV5-hRKp.RPGR demonstrated an anticipated and manageable AE profile through 52 weeks. Safety and efficacy findings support investigation in a phase 3 trial.

Characterization of Retinal Function Using Microperimetry-Derived Metrics in Both Adults and Children With RPGR-Associated Retinopathy.

PURPOSE: To investigate microperimetry testing of retinitis pigmentosa GTPase regulator gene (RPGR)-associated retinopathy in a cohort of children and adults. DESIGN: Prospective observational case series. METHODS: The coefficient of repeatability and intraclass correlation coefficient (ICC) of mean sensitivity (MS) were calculated for mesopic microperimetry. Best-corrected visual acuity (BCVA), contrast sensitivity (CS), MS, total volume (VTOT), and central 3-degree field volume (V3) from volumetric and topographic analyses were acquired. RESULTS: The study recruited 76 individuals with RPGR (53 adults, 23 children). The mean follow-up period was 2.8 years. The ICC values for MS, VTOT, and V3 were 0.982 dB (95% CI, 0.969-0.989 dB), 0.970 dB-steradian (sr) (95% CI, -0.02658 to 0.03691 dB-sr), and 0.986 dB-sr (95% CI, 0.978-0.991), respectively. The r values for interocular MS, VTOT, and V3 were 0.97 (P < .01), 0.97 (P < .01), and 0.98 (P < .01), respectively, indicating strong interocular correlation. The interocular correlation of progression for MS, VTOT, and V3 was 0.81 (P < .01), 0.64 (P < .01), and 0.81 (P < .01), respectively. There was no statistically significant difference in the interocular progression rates for MS or VTOT. V3 did show a statistically significant difference. Most patients lost retinal sensitivity rapidly during their second and third decades of life. CONCLUSIONS: The high degree of reproducibility of results and the good interocular correlation lends this method to accurately monitoring disease progression, as well as supporting validation of the use of MP in assessing the outcomes of gene therapy clinical treatment trials.

A Quantized Representation of Intertemporal Choice in the Brain.

Value [4][5] is typically modeled using a continuous representation (i.e., a Real number). A discrete representation of value has recently been postulated [6]. A quantized representation of probability in the brain was also posited and supported by experimental data [7]. Value and probability are inter-related via Prospect Theory [4][5]. In this paper, we hypothesize that intertemporal choices may also be quantized. For example, people may treat (or discount) 16 days indifferently to 17 days. To test this, we analyzed an intertemporal task by using 2 novel models: quantized hyperbolic discounting, and quantized exponential discounting. Our work here is a re-examination of the behavioral data previously collected for an fMRI study [8]. Both quantized hyperbolic and quantized exponential models were compared using AIC and BIC tests. We found that 13/20 participants were best fit to the quantized exponential model, while the remaining 7/20 were best fit to the quantized hyperbolic model. Overall, 15/20 participants were best fit to models with a 5-bit precision (i.e., 25 = 32 steps). In conclusion, regardless of hyperbolic or exponential, quantized versions of these models are better fit to the experimental data than their continuous forms. We finally outline some potential applications of our findings.

Characteristics Associated with Variation in Corticosteroid Exposure in Children with Steroid-Sensitive Nephrotic Syndrome: Results from a Canadian Longitudinal Study.

Background: Variation in dose and duration of corticosteroids for childhood-onset steroid-sensitive nephrotic syndrome occurs worldwide, likely reflecting the evolving evidence on optimal dosing and variable severity of the disease observed between patients. We conducted a study to determine the associations between site, physician, and patient factors, and average daily corticosteroid dose and duration of therapy. Methods: Data were derived from the Canadian Childhood Nephrotic Syndrome (CHILDNEPH) Project, an observational longitudinal study from 2013 to 2019 of children with nephrotic syndrome involving pediatric nephrologists in 11 sites across Canada. The primary outcome was average daily corticosteroid dose prescribed per episode of proteinuria, reported as mg/m2 prednisone equivalents. Secondary outcome was duration of treatment for each episode of proteinuria in days. Exposure variables were categorized into site-, physician-, and patient-level variables. Results: In total, 328 children, median age at enrollment of 4.3 years old (interquartile range [IQR], 3.6), participated and were followed for a median time of 2.62 years (IQR, 2.6). The observed variability in average daily corticosteroid dose and in duration of therapy was mostly attributed to the site where the patient was treated. Accounting for between patient, physician, and site differences, average daily corticosteroid dose decreased with increasing age (beta coefficient, -0.07; 95% confidence interval [95% CI], -0.09 to -0.05], P<0.001). African and Indigenous ethnicity was associated with longer treatment duration compared with White patients (beta coefficient: African, 42.29, 95% CI, 7.85 to 76.73, P=0.02; Indigenous, 29.65, 95% CI, 2.79 to 56.52, P=0.03). Conclusions: We found practice variation with respect to corticosteroid prescriptions across 11 Canadian sites, and that variation is mostly explained at the site level. Age and ethnicity are important factors to be considered, because they are significantly associated with the average corticosteroid dose and duration of therapy.

A Quantized Representation of Probability in the Brain.

Conventional and current wisdom assumes that the brain represents probability as a continuous number to many decimal places. This assumption seems implausible given finite and scarce resources in the brain. Quantization is an information encoding process whereby a continuous quantity is systematically divided into a finite number of possible categories. Rounding is a simple example of quantization. We apply this information theoretic concept to develop a novel quantized (i.e., discrete) probability distortion function. We develop three conjunction probability gambling tasks to look for evidence of quantized probability representations in the brain. We hypothesize that certain ranges of probability will be lumped together in the same indifferent category if a quantized representation exists. For example, two distinct probabilities such as 0.57 and 0.585 may be treated indifferently. Our extensive data analysis has found strong evidence to support such a quantized representation: 59/76 participants (i.e., 78%) demonstrated a best fit to 4-bit quantized models instead of continuous models. This observation is the major development and novelty of the present work. The brain is very likely to be employing a quantized representation of probability. This discovery demonstrates a major precision limitation of the brain's representational and decision-making ability.

CELLULAR IMAGING OF THE TAPETAL-LIKE REFLEX IN CARRIERS OF RPGR-ASSOCIATED RETINOPATHY.

PURPOSE: To examine the features of the tapetal-like reflex (TLR) in female carriers of RPGR-associated retinopathy by means of adaptive optics scanning light ophthalmoscopy (AOSLO) and spectral domain optical coherence tomography. METHODS: Nine molecularly confirmed RPGR carriers and three healthy controls underwent ocular examination and the following retinal imaging modalities: color photography, near-infrared reflectance, fundus autofluorescence, spectral domain optical coherence tomography, and AOSLO. After identifying TLR areas across all imaging modalities, normalized local contrast of outer retinal bands on spectral domain optical coherence tomography was calculated and AOSLO-acquired photoreceptor mosaic analysis was performed. RESULTS: Seven carriers had TLR areas, which colocalized with increased rod photoreceptor reflectivity on confocal AOSLO and reduced cone photoreceptor densities. Parafoveal TLR areas also exhibited reduced local contrast (i.e., increased reflectivity) of the outer retinal bands on spectral domain optical coherence tomography (inner segment ellipsoid zone and outer segment interdigitation zone). Healthy controls did not show TLR. CONCLUSION: The cellular resolution provided by AOSLO affords the characterization of the photoreceptor mosaic in RPGR carriers with a TLR. Features revealed include reduced cone density, increased cone inner segment diameter, and increased rod outer segment reflectivity.

Natural History Study of Retinal Structure, Progression, and Symmetry Using Ellipzoid Zone Metrics in RPGR-Associated Retinopathy.

PURPOSE: This is a quantitative study of retinal structure, progression rates, and interocular symmetry in retinitis pigmentosa GTPase regulator gene (RPGR)-associated retinopathy using spectral-domain optical coherence tomography (OCT). DESIGN: Prospective, observational cohort study. METHODS: Thirty-eight subjects at Moorfields Eye Hospital in London were assessed with 2 spectral-domain OCT-derived ellipzoid zone (EZ) metrics with repeatability assessments. EZ width (EZW) measurements were made on transfoveal line scans. En face images of the EZ area (EZA) were generated from high-density macular volume scans and were quantified. Baseline size, progression rate, symmetry, associations with age and genotype, and baseline structure-function correlation were investigated. RESULTS: Baseline EZW and EZA measurements were 1963.6 µm and 3.70 mm2, respectively. The mean EZW progression rate was 233.6 µm per year, and the mean EZA rate was 0.67 mm2 per year. Relative interocular difference as an index of symmetry was 3% for both metrics, indicating good baseline symmetry in general-although significant variation existed across the cohort. Analysis of variance found a significant effect of age but not genotype on EZ dimension and progression rates. Larger EZ dimension and greater progression were seen in younger subjects. A positive correlation between EZ dimension and progression was evident. Overall exponential decline rates of 8.2% with EZW and 15.5% with EZA were obtained. Good functional correlation was found with EZW demonstrating stronger correlation; however, EZA correlation with function was also significant. CONCLUSIONS: EZ metrics are sensitive structural biomarkers for measuring residual extent and progression in RPGR-associated retinopathy. Our elucidation of the natural history will provide clinicians and patients with more knowledge about the condition and inform the design and interpretation of interventional trials.

Characterization of Visual Function, Interocular Variability and Progression Using Static Perimetry-Derived Metrics in RPGR-Associated Retinopathy.

Purpose: To characterize bilateral visual function, interocular variability and progression by using static perimetry-derived volumetric and pointwise metrics in subjects with retinitis pigmentosa associated with mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. Methods: This was a prospective longitudinal observational study of 47 genetically confirmed subjects. Visual function was assessed with ETDRS and Pelli-Robson charts; and Octopus 900 static perimetry using a customized, radially oriented 185-point grid. Three-dimensional hill-of-vision topographic models were produced and interrogated with the Visual Field Modeling and Analysis software to obtain three volumetric metrics: VTotal, V30, and V5. These were analyzed together with Octopus mean sensitivity values. Interocular differences were assessed with the Bland-Altman method. Metric-specific exponential decline rates were calculated. Results: Baseline symmetry was demonstrated by relative interocular difference values of 1% for VTotal and 8% with V30. Degree of symmetry varied between subjects and was quantified with the subject percentage interocular difference (SPID). SPID was 16% for VTotal and 17% for V30. Interocular symmetry in progression was greatest when quantified by VTotal and V30, with 73% and 64% of subjects possessing interocular rate differences smaller in magnitude than respective annual progression rates. Functional decline was evident with increasing age. An overall annual exponential decline of 6% was evident with both VTotal and V30. Conclusions: In general, good interocular symmetry exists; however, there was both variation between subjects and with the use of various metrics. Our findings will guide patient selection and design of RPGR treatment trials, and provide clinicians with specific prognostic information to offer patients affected by this condition.

QUANTITATIVE ANALYSIS OF HYPERAUTOFLUORESCENT RINGS TO CHARACTERIZE THE NATURAL HISTORY AND PROGRESSION IN RPGR-ASSOCIATED RETINOPATHY.

PURPOSE: Quantitative analysis of hyperautofluorescent rings and progression in subjects with retinitis pigmentosa associated with retinitis pigmentosa GTPase regulator (RPGR) gene mutations. METHODS: Prospective observational study of 46 subjects. Ring area, horizontal and vertical diameter measurements taken from outer and inner ring borders. Intraobserver repeatability, baseline measurements, progression rates, interocular symmetry, and association with age and genotype were investigated. RESULTS: Baseline ring area was 11.8 ± 13.4 mm and 11.4 ± 13.2 mm for right and left eyes, respectively, with very strong interocular correlation (r = 0.9398; P < 0.0001). Ring area constriction was 1.5 ± 2.0 mm/year and 1.3 ± 1.9 mm/year for right and left eyes, respectively, with very strong interocular correlation (r = 0.878, P < 0.0001). Baseline ring area and constriction rate correlated negatively with age (r = -0.767; P < 0.0001 and r = -0.644, P < 0.0001, respectively). Constriction rate correlated strongly with baseline area (r = 0.850, P < 0.0001). Age, but not genotype, exerted a significant effect on constriction rates (P < 0.0001), with greatest rates of progression seen in younger subjects. An exponential decline overall was found. CONCLUSION: This study provides disease-specific baseline values and progression rates together with a repeatability assessment of fundus autofluorescence metrics. Our findings can guide future treatment trials and contribute to the clinical care of patients with RPGR-associated retinitis pigmentosa.

Reliability and Repeatability of Cone Density Measurements in Patients With Stargardt Disease and RPGR-Associated Retinopathy.

Purpose: To assess reliability and repeatability of cone density measurements by using confocal and (nonconfocal) split-detector adaptive optics scanning light ophthalmoscopy (AOSLO) imaging. It will be determined whether cone density values are significantly different between modalities in Stargardt disease (STGD) and retinitis pigmentosa GTPase regulator (RPGR)-associated retinopathy. Methods: Twelve patients with STGD (aged 9-52 years) and eight with RPGR-associated retinopathy (aged 11-31 years) were imaged using both confocal and split-detector AOSLO simultaneously. Four graders manually identified cone locations in each image that were used to calculate local densities. Each imaging modality was evaluated independently. The data set consisted of 1584 assessments of 99 STGD images (each image in two modalities and four graders who graded each image twice) and 928 RPGR assessments of 58 images (each image in two modalities and four graders who graded each image twice). Results: For STGD assessments the reliability for confocal and split-detector AOSLO was 67.9% and 95.9%, respectively, and the repeatability was 71.2% and 97.3%, respectively. The differences in the measured cone density values between modalities were statistically significant for one grader. For RPGR assessments the reliability for confocal and split-detector AOSLO was 22.1% and 88.5%, respectively, and repeatability was 63.2% and 94.5%, respectively. The differences in cone density between modalities were statistically significant for all graders. Conclusions: Split-detector AOSLO greatly improved the reliability and repeatability of cone density measurements in both disorders and will be valuable for natural history studies and clinical trials using AOSLO. However, it appears that these indices may be disease dependent, implying the need for similar investigations in other conditions.

Quantitative Analysis of Retinal Structure Using Spectral-Domain Optical Coherence Tomography in RPGR-Associated Retinopathy.

PURPOSE: To quantify retinal structure and progression using spectral-domain optical coherence tomography (SDOCT) in patients with retinitis pigmentosa (RP) associated with retinitis pigmentosa GTPase regulator gene (RPGR) mutations. DESIGN: Retrospective observational case series. METHODS: Setting: Moorfields Eye Hospital, London, United Kingdom. SUBJECTS: Both eyes of 32 patients. SDOCT follow-up period of >1 year (3.1 ± 1.4 years). MAIN OUTCOME MEASURES: Ellipsoid zone (EZ) width (EZW) and outer nuclear layer (ONL) and inner retinal layer (IRL) thickness measurements. Progression rates, interocular symmetry, and association with age and genotype were investigated. RESULTS: Significant differences were observed between baseline and final measurements of EZW and ONL thickness, but not for IRL thickness. Baseline and final EZWs were 2438 ± 1646 µm and 1901 ± 1423 µm for right eyes (P < .0001); 2420 ± 1758 µm and 1922 ± 1482 µm for left eyes (P < .0001). EZW constriction rates were 176.6 ± 130.1 µm/year and 173.1 ± 146.8 µm/year for right and left eyes. ONL thinning rates were 2.58 ± 2.85 µm/year and 2.52 ± 3.54 µm/year for right and left eyes. Interocular differences in EZW and ONL progression were not significant (P = .8609 and P = .6735, respectively). Strong correlations were found between EZW constriction rates of right and left eyes (rs = 0.627, P = .0002) and between EZW constriction and baseline EZW (rs = 0.714, P < .0001). There was moderate negative correlation between EZW constriction and age (rs = -0.532, P < .0001). Correlation between ONL thinning and age was not significant, as were differences between EZW and ONL progression rates with respect to genotype. CONCLUSIONS: This study provides SDOCT progression rates for RPGR-associated RP. There is overall interocular symmetry with implications for future treatment trials where 1 eye could serve as a control.

Duration of intraocular gases following vitreoretinal surgery.

BACKGROUND: Intraocular gas tamponades are an important tool in modern vitreoretinal surgery. However, there is considerable variation in their use and perceptions amongst clinicians regarding these agents. METHODS: An electronic survey of vitreoretinal surgeons in the UK was undertaken to establish the patterns of use and surgeons' estimates of the longevity and expansion timing of gas tamponades. In addition, data were prospectively collected on the longevity of gas tamponades in 114 patients from our unit. An analysis was performed to identify patient or surgery factors affecting gas longevity RESULTS: A wide variation in the patterns of use and estimates of longevity and expansion timing of intraocular tamponades was found in the survey of vitreoretinal surgeons. Data from our unit give informed estimates on the longevity of three commonly used tamponades. For 30 % sulphur hexafluoride (SF6), mean 18.0 days, standard deviation (SD) 2.6 days. For 20 % hexafluoroethane (C2F6), mean 34.5 days, SD 3.3 days. For 15 % perfluoropropane (C3F8), mean 67.7 days SD 5.5 days. In the C2F6 group there was correlation between longer duration of the gas bubble and longer axial length (r = 0.438, p = 0.02) and longer gas duration with male sex (p = 0.002). CONCLUSIONS: We present informed gas tamponade longevity figures in clinical practice and report statistically significant associations between longer gas longevity and increasing axial length and male sex.

Cone Photoreceptor Structure in Patients With X-Linked Cone Dysfunction and Red-Green Color Vision Deficiency.

PURPOSE: Mutations in the coding sequence of the L and M opsin genes are often associated with X-linked cone dysfunction (such as Bornholm Eye Disease, BED), though the exact color vision phenotype associated with these disorders is variable. We examined individuals with L/M opsin gene mutations to clarify the link between color vision deficiency and cone dysfunction. METHODS: We recruited 17 males for imaging. The thickness and integrity of the photoreceptor layers were evaluated using spectral-domain optical coherence tomography. Cone density was measured using high-resolution images of the cone mosaic obtained with adaptive optics scanning light ophthalmoscopy. The L/M opsin gene array was characterized in 16 subjects, including at least one subject from each family. RESULTS: There were six subjects with the LVAVA haplotype encoded by exon 3, seven with LIAVA, two with the Cys203Arg mutation encoded by exon 4, and two with a novel insertion in exon 2. Foveal cone structure and retinal thickness was disrupted to a variable degree, even among related individuals with the same L/M array. CONCLUSIONS: Our findings provide a direct link between disruption of the cone mosaic and L/M opsin variants. We hypothesize that, in addition to large phenotypic differences between different L/M opsin variants, the ratio of expression of first versus downstream genes in the L/M array contributes to phenotypic diversity. While the L/M opsin mutations underlie the cone dysfunction in all of the subjects tested, the color vision defect can be caused either by the same mutation or a gene rearrangement at the same locus.

The Effect of Multispot Laser Panretinal Photocoagulation on Retinal Sensitivity and Driving Eligibility in Patients With Diabetic Retinopathy.

IMPORTANCE: Panretinal photocoagulation (PRP) for proliferative diabetic retinopathy (PDR) may lead to peripheral field loss that prevents driving. Anti-vascular endothelial growth factor agents are proposed as treatments for PDR that spare peripheral vision. If multispot lasers cause less visual field loss, continuing to perform PRP may be justified. OBJECTIVE: To assess the effect of bilateral multispot laser PRP on retinal sensitivity and driving visual fields in PDR. DESIGN, SETTING, AND PARTICIPANTS: This prospective nonrandomized interventional cohort analysis performed at a tertiary referral center included 43 laser-naive patients with PDR that required bilateral PRP. Participants were recruited from June 27, 2012, to October 14, 2013. At baseline and 6-month follow-up, patients underwent detailed static and kinetic perimetry, microperimetry, optical coherence tomography, wide-field color fundus photography, and fluorescein angiography. Quantitative change in retinal sensitivity was assessed by comparing the mean global retinal sensitivity before and after laser treatment and by comparing the modeled hill of vision by deriving a volumetric measure. Final follow-up was completed on May 21, 2014. INTERVENTIONS: Multispot laser treatment was applied using standard parameters, until neovascularization regressed or complete retinal coverage was achieved. MAIN OUTCOMES AND MEASURES: Participants who passed the Esterman binocular visual field test for driving in the United Kingdom (at least 120° horizontal field with no significant defects within the central 20°) and full-field and macular retinal sensitivity. RESULTS: Of the 43 patients (17 men; 26 women; mean [SD] age, 46.6 [13.3] years), 38 (88%) completed the study. Before treatment, 41 of 43 patients (95%) passed the Esterman visual field test for driving; after completion of laser treatment, 35 of 38 patients (92%) passed. The mean (SD) change in retinal sensitivity on static perimetry was -1.4 (3.7) (95% CI, -2.7 to -0.1) dB OD and -2.4 (2.9) (95% CI, -3.4 to -1.4) dB OS. Mean (SD) 4° macular sensitivity decreased by 3.0 (5.2) dB OD and 2.6 (5.4) dB OS. CONCLUSIONS AND RELEVANCE: This prospective study investigating the effects of multispot laser PRP on retinal sensitivity demonstrates a high likelihood of retaining eligibility to drive based on adequate visual field. A mild loss of retinal sensitivity was detected at 6 months after completion of laser treatment. Further change to visual fields may have occurred with longer follow-up. This study provides information that might be used to counsel patients requiring PRP and informs the debate regarding the role of anti-vascular endothelial growth factor therapy in patients with PDR who might otherwise receive laser treatment.

RPGR-associated retinopathy: clinical features, molecular genetics, animal models and therapeutic options.

Retinitis pigmentosa GTPase regulator (RPGR) gene sequence variants account for the vast majority of X linked retinitis pigmentosa (RP), which is one of the most severe forms of RP. Symptoms of nyctalopia typically begin in childhood, with increasing loss of peripheral visual field during teenage years, and progressive central visual loss during the second to fourth decade of life. There is however marked intrafamilial and interfamilial phenotypic heterogeneity in affected males and carrier females. There is now a far greater understanding of the range of phenotypes associated with variants in this gene; including rod-cone dystrophy, cone-rod dystrophy, cone dystrophy, macular dystrophy and non-ocular phenotypes. There are also increasingly established genotype-phenotype associations and structure-function correlations. RPGR is involved in ciliary function, with ciliary dysfunction now recognised as the mechanism underlying a large proportion of inherited retinal disease. There has been significant progress in identifying naturally occurring animal models and developing novel models to define the underlying disease mechanisms and to test gene replacement therapy, in addition to advances in human retinal imaging, culminating in completed and planned clinical trials. These significant developments will be discussed.

The Canadian Childhood Nephrotic Syndrome (CHILDNEPH) Project: overview of design and methods.

BACKGROUND: Nephrotic syndrome is a commonly acquired kidney disease in children that causes significant morbidity due to recurrent episodes of heavy proteinuria. The management of childhood nephrotic syndrome is known to be highly variable among physicians and care centres. OBJECTIVES: The primary objective of the study is to determine centre-, physician-, and patient-level characteristics associated with steroid exposure and length of steroid treatment. We will also determine the association of dose and duration of steroid treatment and time to first relapse as a secondary aim. An embedded qualitative study utilizing focus groups with health care providers will enrich the quantitative results by providing an understanding of the attitudes, beliefs and local contextual factors driving variation in care. DESIGN: Mixed-methods study; prospective observational cohort (quantitative component), with additional semi-structured focus groups of healthcare professionals (qualitative component). SETTING: National study, comprised of all 13 Canadian pediatric nephrology clinics. PATIENTS: 400 patients under 18 years of age to be recruited over 2.5 years. MEASUREMENTS: Steroid doses for all episodes (first presentation, first and subsequent relapses) tracked over course of the study. Physician and centre-level characteristics catalogued, with reasons for treatment preferences documented during focus groups. METHODS: All patients tracked prospectively over the course of the study, with data comprising a prospective registry. One focus group at each site to enrich understanding of variation in care. LIMITATIONS: Contamination of treatment protocols between physicians may occur as a result of concurrent focus groups. CONCLUSIONS: Quantitative and qualitative results will be integrated at end of study and will collectively inform strategies for the development and implementation of standardized evidence-based protocols across centres.

CONTEXTE: Le syndrome néphrotique est une néphropathie fréquente chez l'enfant, qui cause une morbidité considérable en raison de la récurrence d'épisodes de protéinurie importante. La gestion du syndrome néphrotique de l'enfant varie énormément d'un médecin et d'un centre de soins à l'autre. OBJECTIFS: L'objectif principal de cette étude est de déterminer les caractéristiques associées à l'exposition et la durée du traitement aux stéroïdes, liées au centre, au médecin et au patient. En deuxième lieu, nous déterminerons la corrélation entre la dose et la durée du traitement aux stéroïdes, puis la durée avant la première rechute. À cette étude s'ajoutera une étude qualitative avec des groupes de discussion composés de professionnels de la santé qui viendra enrichir les résultats quantitatifs en favorisant une meilleure compréhension des attitudes, des croyances et des facteurs contextuels locaux qui entraînent des variations dans les soins. TYPE D'ÉTUDE: Une étude méthodologique mixte; étude d'observation de cohorte prospective (composante quantitative), combinée avec des groupes de discussion semi-structurés composés de professionnels de la santé (composante qualitative). CONTEXTE/ÉCHANTILLON: Étude nationale, constituée des 13 cliniques canadiennes de néphrologie pédiatrique. PARTICIPANTS: 400 patients âgés de moins de 18 ans, à recruter sur une période de 2,5 années. MESURES: Suivi des doses de stéroïdes pour chacun des épisodes (première présentation, première rechute et suivantes), tout au long de l'étude. Catalogage des caractéristiques liées au médecin ou au centre, et attestation des raisons justifiant les préférences de traitement au cours des séances avec les groupes de discussion. MÉTHODES: Tous les patients suivis de manière prospective dans le cadre de l'étude, dont les données constituent un registre prospectif. Un groupe de discussion à chaque endroit, afin d'enrichir la compréhension des variations dans les soins. LIMITES DE L'ÉTUDE: La contamination des protocoles de traitement entre les médecins peut se produire en raison de la tenue simultanée de groupes de discussions. CONCLUSIONS: Les résultats quantitatifs et qualitatifs seront intégrés à la fin de l'étude et permettront de mettre en place des stratégies de développement et de mise en œuvre de protocoles normalisés et fondés sur des données probantes.

GDNF-independent ureteric budding: role of PI3K-independent activation of AKT and FOSB/JUN/AP-1 signaling.

A significant fraction of mice deficient in either glial cell-derived neurotrophic factor (GDNF) or its co-receptors (Gfrα1, Ret), undergoes ureteric bud (UB) outgrowth leading to the formation of a rudimentary kidney. Previous studies using the isolated Wolffian duct (WD) culture indicate that activation of fibroblast growth factor (FGF) receptor signaling, together with suppression of BMP/Activin signaling, is critical for GDNF-independent WD budding (Maeshima et al., 2007). By expression analysis of embryonic kidney from Ret((-/-)) mice, we found the upregulation of several FGFs, including FGF7. To examine the intracellular pathways, we then analyzed GDNF-dependent and GDNF-independent budding in the isolated WD culture. In both conditions, Akt activation was found to be important; however, whereas this occurred through PI3-kinase in GDNF-dependent budding, in the case of GDNF-independent budding, Akt activation was apparently via a PI3-kinase independent mechanism. Jnk signaling and the AP-1 transcription factor complex were also implicated in GDNF-independent budding. FosB, a binding partner of c-Jun in the formation of AP-1, was the most highly upregulated gene in the ret knockout kidney (in which budding had still occurred), and we found that its siRNA-mediated knockdown in isolated WDs also blocked GDNF-independent budding. Taken together with the finding that inhibition of Jnk signaling does not block Akt activation/phosphorylation in GDNF-independent budding, the data support necessary roles for both FosB/Jun/AP-1 signaling and PI3-kinase-independent activation of Akt in GDNF-independent budding. A model is proposed for signaling events that involve Akt and JNK working to regulate GDNF-independent WD budding.

Hepatocyte nuclear factors 4α and 1α regulate kidney developmental expression of drug-metabolizing enzymes and drug transporters.

The transcriptional regulation of drug-metabolizing enzymes and transporters (here collectively referred to as DMEs) in the developing proximal tubule (PT) is not well understood. As in the liver, DME regulation in the PT may be mediated through nuclear receptors, which are thought to "sense" deviations from homeostasis by being activated by ligands, some of which are handled by DMEs, including drug transporters. Systems analysis of transcriptomic data during kidney development predicted a set of upstream transcription factors, including hepatocyte nuclear factor 4α (Hnf4a) and Hnf1a, as well as Nr3c1 (Gr), Nfe2l2 (Nrf2), peroxisome proliferator-activated receptor α (Pparα), and Tp53. Motif analysis of cis-regulatory enhancers further suggested that Hnf4a and Hnf1a are the main transcriptional regulators of DMEs in the PT. Available expression data from tissue-specific Hnf4a knockout tissues revealed that distinct subsets of DMEs were regulated by Hnf4a in a tissue-specific manner. Chromatin immunoprecipitation combined with massively parallel DNA sequencing was performed to characterize the PT-specific binding sites of Hnf4a in rat kidneys at three developmental stages (prenatal, immature, adult), which further supported a major role for Hnf4a in regulating PT gene expression, including DMEs. In ex vivo kidney organ culture, an antagonist of Hnf4a (but not a similar inactive compound) led to predicted changes in DME expression, including among others Fmo1, Cyp2d2, Cyp2d4, Nqo2, as well as organic cation transporters and organic anion transporters Slc22a1 (Oct1), Slc22a2 (Oct2), Slc22a6 (Oat1), Slc22a8 (Oat3), and Slc47a1 (Mate1). Conversely, overexpression of Hnf1a and Hnf4a in primary mouse embryonic fibroblasts, sometimes considered a surrogate for mesenchymal stem cells, induced expression of several of these proximal tubule DMEs, as well as epithelial markers and a PT-enriched brush border marker Ggt1. These cells had organic anion transporter function. Taken together, the data strongly supports a critical role for HNF4a and Hnf1a in the tissue-specific regulation of drug handling and differentiation toward a PT-like cellular identity. We discuss our data in the context of the "remote sensing and signaling hypothesis" (Ahn and Nigam, 2009; Wu et al., 2011).