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Craig T.A.,Nephrology and Hypertension Research | Kumar R.,Nephrology and Hypertension Research | Kumar R.,Mayo Medical School
Biochemical and Biophysical Research Communications | Year: 2010

To gain insights into the mechanism of action of sclerostin, a protein that regulates bone mass, we performed yeast two-hybrid analyses using human SOST (sclerostin) cDNA cloned into pGBKT7 DNA-binding domain vector as a bait, and a normalized, high-complexity, universal cDNA library in a GAL4 activating domain vector. We identified an interaction between sclerostin and the carboxyl-terminal portion of the receptor tyrosine-protein kinase erbB-3. To determine the biological relevance of this interaction, we treated MC3T3-E1 mouse osteoblast cells transfected with either a SOST expression plasmid or a control vector, with recombinant heregulin/neuregulin. Phospho-p44/42 (Thr202/Tyr204) MAPK was assessed in heregulin/neuregulin treated cells. We observed an increase in phospho-p44/42 (Thr202/Tyr204) MAPK concentrations in SOST transfected cells but not in cells transfected with a control vector, thus demonstrating a modulatory effect of sclerostin on heregulin/neuregulin signaling in osteoblasts. The data demonstrate that sclerostin functions in part, by modulating the activity of erbB-3. © 2010 Elsevier Inc.

Craig T.A.,Nephrology and Hypertension Research | Zhang Y.,Mayo Medical School | Magis A.T.,University of Illinois at Urbana - Champaign | Funk C.C.,Institute for Systems Biology | And 4 more authors.
Zebrafish | Year: 2014

The sterol hormone, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), regulates gene expression and messenger RNA (mRNA) concentrations in zebrafish in vivo. Since mRNA concentrations and translation are influenced by micro-RNAs (miRNAs), we examined the influence of 1α,25(OH)2D3 on miRNA expression in zebrafish in vivo with whole transcriptome RNA sequencing, searched for miRNA binding sites in 1α,25(OH)2D 3-sensitive genes, and performed correlation analyses between 1α,25(OH)2D3-sensitive miRNAs and mRNAs. In vehicle-and 1α,25(OH)2D3-treated, 7-day postfertilization larvae, between 282 and 295 known precursor miRNAs were expressed, and in vehicle-and 1α,25(OH)2D3-treated fish, between 83 and 122 novel miRNAs were detected. Following 1α,25(OH)2D3 treatment, 31 precursor miRNAs were differentially expressed (p<0.05). The differentially expressed miRNAs are predicted to potentially alter mRNAs for metabolic enzymes, transcription factors, growth factors, and Jak-STAT signaling. We verified the role of a 1α,25(OH)2D3-sensitive miRNA, miR125b, by demonstrating alterations in the concentrations of the mRNA of a 1α,25(OH)2D3-regulated gene, Cyp24a1, following transfection of renal cells with a miR125b miRNA mimic. Changes in the Cyp24a1 mRNA concentration by the miR125b miRNA mimic were associated with changes in the protein for Cyp24a1. Our data show that 1α,25(OH)2D 3 regulates miRNA in zebrafish larvae in vivo and could thereby influence vitamin D-sensitive mRNA concentrations. © 2014 Mary Ann Liebert, Inc.

Hogan M.C.,Mayo Medical School | Lieske J.C.,Mayo Medical School | Lienczewski C.C.,University of Michigan | Nesbitt L.L.,Cardiovascular Research | And 14 more authors.
BMC Nephrology | Year: 2015

Background: Glomerular diseases are potentially fatal, requiring aggressive interventions and close monitoring. Urine is a readily-accessible body fluid enriched in molecular signatures from the kidney and therefore particularly suited for routine clinical analysis as well as development of non-invasive biomarkers for glomerular diseases. Methods: The Nephrotic Syndrome Study Network (NEPTUNE; ClinicalTrials.gov Identifier NCT01209000) is a North American multicenter collaborative consortium established to develop a translational research infrastructure for nephrotic syndrome. This includes standardized urine collections across all participating centers for the purpose of discovering non-invasive biomarkers for patients with nephrotic syndrome due to minimal change disease, focal segmental glomerulosclerosis, and membranous nephropathy. Here we describe the organization and methods of urine procurement and banking procedures in NEPTUNE. Results: We discuss the rationale for urine collection and storage conditions, and demonstrate the performance of three experimental analytes (neutrophil gelatinase-associated lipocalin [NGAL], retinol binding globulin, and alpha-1 microglobulin) under these conditions with and without urine preservatives (thymol, toluene, and boric acid). We also demonstrate the quality of RNA and protein collected from the urine cellular pellet and exosomes. Conclusions: The urine collection protocol in NEPTUNE allows robust detection of a wide range of proteins and RNAs from urine supernatant and pellets collected longitudinally from each patient over 5 years. Combined with the detailed clinical and histopathologic data, this provides a unique resource for exploration and validation of new or accepted markers of glomerular diseases. Trial registration: ClinicalTrials.gov Identifier NCT01209000 © 2015 Hogan et al.

Craig T.A.,Nephrology and Hypertension Research | Zhang Y.,Mayo Medical School | McNulty M.S.,Nephrology and Hypertension Research | Middha S.,Mayo Medical School | And 8 more authors.
Molecular Endocrinology | Year: 2012

The biological role of vitamin D receptors (VDR), which are abundantly expressed in developing zebrafish (Danio rerio) as early as 48 h after fertilization, and before the development of a mineralized skeleton and mature intestine and kidney, is unknown. We probed the role of VDR in developing zebrafish biology by examining changes in expression of RNA by whole transcriptome shotgun sequencing (RNA-seq) in fish treated with picomolar concentrations of the VDR ligand and hormonal form of vitamin D3, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3)].We observed significant changes in RNAs of transcription factors, leptin, peptide hormones, and RNAs encoding proteins of fatty acid, amino acid, xenobiotic metabolism, receptor-activator of NFκB ligand (RANKL), and calcitonin-like ligand receptor pathways. Early highly restricted, and subsequent massive changes in more than 10% of expressed cellular RNA were observed. At days post fertilization (dpf) 2 [24 h 1α,25(OH)2D3-treatment], only four RNAs were differentially expressed (hormone vs. vehicle). On dpf 4 (72 h treatment), 77 RNAs; on dpf 6 (120 h treatment) 1039 RNAs; and on dpf 7 (144 h treatment), 2407 RNAs were differentially expressed in response to 1α,25(OH)2D3. Fewer RNAs (n = 481) were altered in dpf 7 larvae treated for 24 h with 1α,25(OH)2D3 vs. those treated with hormone for 144 h. At dpf 7, in 1α,25(OH)2D3-treated larvae, pharyngeal cartilage was larger and mineralization was greater. Changes in expression of RNAs for transcription factors, peptide hormones, and RNAs encoding proteins integral to fatty acid, amino acid, leptin, calcitonin-like ligand receptor, RANKL, and xenobiotic metabolism pathways, demonstrate heretofore unrecognized mechanisms by which 1α,25(OH)2D3 functions in vivo in developing eukaryotes. © 2012 by The Endocrine Society.

Thatava T.,Mayo Medical School | Armstrong A.S.,Mayo Medical School | De Lamo J.,Mayo Medical School | Edukulla R.,Nephrology and Hypertension Research | And 7 more authors.
Stem Cell Research and Therapy | Year: 2011

Introduction. End-stage renal disease (ESRD) is a major public health problem. Although kidney transplantation is a viable therapeutic option, this therapy is associated with significant limitations, including a shortage of donor organs. Induced pluripotent stem (iPS) cell technology, which allows derivation of patient-specific pluripotent stem cells, could provide a possible alternative modality for kidney replacement therapy for patients with ESRD. Methods. The feasibility of iPS cell generation from patients with a history of ESRD was investigated using lentiviral vectors expressing pluripotency- associated factors. Results: In the present article we report, for the first time, generation of iPS cells from kidney transplant recipients with a history of autosomal-dominant polycystic kidney disease (ADPKD), systemic lupus erythematosus, or Wilms tumor and ESRD. Lentiviral transduction of OCT4, SOX2, KLF4 and c-MYC, under feeder-free conditions, resulted in reprogramming of skin-derived keratinocytes. Keratinocyte-derived iPS cells exhibited properties of human embryonic stem cells, including morphology, growth properties, expression of pluripotency genes and surface markers, spontaneous differentiation and teratoma formation. All iPS cell clones from the ADPKD patient retained the conserved W3842X mutation in exon 41 of the PKD1 gene. Conclusions: Our results demonstrate successful iPS cell generation from patients with a history of ESRD, PKD1 gene mutation, or chronic immunosuppression. iPS cells from autosomal kidney diseases, such as ADPKD, would provide unique opportunities to study patient-specific disease pathogenesis in vitro. © 2011 Thatava et al.; licensee BioMed Central Ltd.

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