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BACKGROUND: Tolvaptan slows expansion of kidney volume and kidney function decline in adults with autosomal dominant polycystic kidney disease (ADPKD). Progression during childhood could be treated before irreversible kidney damage occurs, but trial data are lacking. We evaluated the safety and efficacy of tolvaptan in children/adolescents with ADPKD. METHODS: This was the 1-year, randomized, double-blind, portion of a phase 3b, two-part trial being conducted at 20 academic pediatric nephrology centers. Key eligibility criteria were ADPKD and eGFR ≥60 ml/min per 1.73 m2. Participants aged 12-17 years were the target group (group 1, enrollment goal n≥60); participants aged 4-11 years could additionally enroll (group 2, anticipated enrollment approximately 40). Treatments were tolvaptan or placebo titrated by body weight and tolerability. Coprimary end points, change from baseline in spot urine osmolality and specific gravity at week 1, assessed inhibition of antidiuretic hormone activity. The key secondary end point was change in height-adjusted total kidney volume (htTKV) to month 12 in group 1. Additional end points were safety/tolerability and quality of life. Statistical comparisons were exploratory and post hoc. RESULTS: Among the 91 randomized (group 1, n=66; group 2, n=25), least squares (LS) mean reduction (±SEM) in spot urine osmolality at week 1 was greater with tolvaptan (-390 [28] mOsm/kg) than placebo (-90 [29] mOsm/kg; P0.05). For tolvaptan and placebo, respectively, 65% and 16% of subjects experienced aquaretic adverse events, and 2% and 0% experienced hypernatremia. There were no elevated transaminases or drug-induced liver injuries. Four participants discontinued tolvaptan, and three discontinued placebo. Quality-of-life assessments remained stable. CONCLUSIONS: Tolvaptan exhibited pharmacodynamic activity in pediatric ADPKD. Aquaretic effects were manageable, with few discontinuations. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Safety, Pharmacokinetics, Tolerability and Efficacy of Tolvaptan in Children and Adolescents With ADPKD (Autosomal Dominant Polycystic Kidney Disease) NCT02964273. ispartof: CLINICAL JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY vol:18 issue:1 pages:36-46 ispartof: location:United States status: published

Transcription factor Ap2b (TFAP2B), an AP-2 family transcription factor, binds to the palindromic consensus DNA sequence, 5′-GCCN3-5GGC-3’. Mice lacking functional Tfap2b gene die in the perinatal or neonatal period with cystic dilatation of the kidney distal tubules and collecting ducts, a phenotype resembling autosomal recessive polycystic kidney disease (ARPKD). Human ARPKD is caused by mutations in PKHD1, DZIP1L, and CYS1, which are conserved in mammals. In this study, we examined the potential role of TFAP2B as a common regulator of Pkhd1 and Cys1. We determined the transcription start site (TSS) of Cys1 using 5′ Rapid Amplification of cDNA Ends (5′RACE); the TSS of Pkhd1 has been previously established. Bioinformatic approaches identified cis-regulatory elements, including two TFAP2B consensus binding sites, in the upstream regulatory regions of both Pkhd1 and Cys1. Based on reporter gene assays performed in mouse renal collecting duct cells (mIMCD-3), TFAP2B activated the Pkhd1 and Cys1 promoters and electromobility shift assay (EMSA) confirmed TFAP2B binding to the in silico identified sites. These results suggest that Tfap2b participates in a renal epithelial cell gene regulatory network that includes Pkhd1 and Cys1. Disruption of this network impairs renal tubular differentiation, causing ductal dilatation that is the hallmark of recessive PKD.

Additional file 3: Supplementary Methods. Protocol for determination of whether patient satisfied Cleveland Clinic criteria.

Additional file 4: Table S1. Billing Codes from ICD-9 and ICD-10 for Cleveland Clinical criteria for PHTS.

Single particle electron microscopy (EM) allows us to examine the molecular world and gain insights into protein structures implicated in human disease. Visualizing the 3D architecture of the macromolecules can inform drug design and preventative care. While X-ray crystallography and NMR are able to resolve atomic structures, the methodology is better suited for smaller structures with limited flexibility. Single particle EM allows us analyze larger structures that have inherent flexibility. Protein structures can broadly be categorized as symmetry or asymmetric. There are computational advantages when analyzing symmetrical structures. Specifically, structural information can be extrapolated from fewer vantage points. Thus, symmetrical macromolecules are an advantageous for pioneering new methodologies in single particle EM. Rotavirus double layered particles (DLPs) are large macromolecular complexes that display icosahedral symmetry. Previous studies have led to a high resolution structure of transcriptionally inactive rotavirus frozen in time. However, to more fully understand rotavirus we need to examine the structure under transcriptionally active conditions. To expand our understanding, we first evaluated these viral assemblies using cryo-EM under active and inactive conditions. We found both internal and external structural differences. Based on these findings we sought to further our understanding of these nano-machines by developing a liquid cell environment to evaluate their dynamics over time. Our research not only developed a new methodology to evaluate active particles over time, we also found that the mobility of the DLPs were directly correlated to the level of transcriptional activity. When analyzing asymmetrical and flexible protein complexes previous studies have utilized methodologies to limit the proteins' conformational variability. While this does allow for a higher resolution structure, it limits our understanding to a specific orientation and compromises the biological insights. BRCA1 is an asymmetric protein containing a large flexible region and is important in the prevention of breast cancer. We utilize silicon nitride microchips with integrated wells and decorated with a lipid monolayer to capture and image BRCA1 complexes. This imaging platform minimizes heterogeneity and ensures the sample quality while not biasing confirmation. Thus, allowing for high resolution cryo-EM imaging of flexible native proteins. We were able to examine BRCA1 complexes from cells at both the primary and metastatic sites. Our ability to visualize these proteins in their native form provide insights into the variability of BRCA1 in disease progression. We found that BRCA1 complexes isolated from metastatic cells have additional density in the C-terminal domain. Our data suggests this density it due an interaction with p53. Overall, our methodologies highlight the power of single particle EM for studying protein complexes. Furthermore, our findings emphasize the importance of examining protein complexes in their native state. PHD Single particle electron microscopy (EM) allows us to examine the molecular world and gain insights into protein structures implicated in human disease. Visualizing the 3D architecture of macromolecules can inform drug design and preventative care. While X-ray crystallography and NMR are able to resolve atomic structures, the methodology is better suited for smaller structures with limited flexibility. Single particle EM allow us analyze larger structures that have inherent flexibility. Protein structures can broadly be categorized as symmetry or asymmetric. There are computational advantages when analyzing symmetrical structures. Specifically, structural information can be extrapolated from fewer vantage points. Thus, symmetrical macromolecules are an advantageous for pioneering new methodologies in single particle EM. Rotavirus double layered particles (DLPs) are large, highly symmetrical macromolecular complexes that represent an ideal model system for developing technology. Previous studies have led to a high resolution structure of inactive rotavirus DLP frozen in time. However, to more fully understand rotavirus we need to examine the structure under active conditions. To expand our understanding, we first evaluated these viral assemblies using cryo-EM under active and inactive conditions. We found structural differences. Based on these findings we sought to further our understanding of these nano-machines by developing a liquid cell environment to evaluate their dynamics over time. Our new methodology revealed new insights into the mobility of the DLPs. When analyzing asymmetrical and flexible protein complexes previous studies have utilized methodologies to limit the proteins’ movement. While this does allow for a higher resolution structure, it limits our understanding to a specific orientation and compromises the biological insights. BRCA1 is a highly flexible asymmetric protein implicated in the development of breast cancer. We utilize specialized microchips to capture and image BRCA1 complexes. This imaging platform ensures sample quality and allows for high resolution cryoEM imaging of flexible native proteins. We were able to examine BRCA1 complexes from cells at both the primary and metastatic sites. Our ability to visualize these proteins in their native form provide insights into the variability of BRCA1 in disease progression. Our data found that BRCA1 complexes isolated from metastatic cells are structurally different than those at the primary site. Overall, our methodologies highlight the power of single particle EM for studying protein complexes. Furthermore, our findings emphasize the importance of examining protein complexes in their native state.