Rapamycin Ameliorates PKD Resulting from Conditional Inactivation of Pkd1
ABSTRACT Aberrant activation of the mammalian target of rapamycin (mTOR) pathway occurs in polycystic kidney disease (PKD). mTOR inhibitors, such as rapamycin, are highly effective in several rodent models of PKD, but these models result from mutations in genes other than Pkd1 and Pkd2, which are the primary genes responsible for human autosomal dominant PKD. To address this limitation, we tested the efficacy of rapamycin in a mouse model that results from conditional inactivation of Pkd1. Mosaic deletion of Pkd1 resulted in PKD and replicated characteristic features of human PKD including aberrant mTOR activation, epithelial proliferation and apoptosis, and progressive fibrosis. Treatment with rapamycin was highly effective: It reduced cyst growth, preserved renal function, inhibited epithelial cell proliferation, increased apoptosis of cyst-lining cells, and inhibited fibrosis. These data provide in vivo evidence that rapamycin is effective in a human-orthologous mouse model of PKD.
Full-textDOI: · Available from: Jonathan M. Shillingford, Ph.D., Sep 28, 2015
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Article: Rapamycin Ameliorates PKD Resulting from Conditional Inactivation of Pkd1
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- "Stained sections were visualized under bright field microscopy and twenty random fields of vision (FOV) in each experimental and control group were captured for quantification. Images were quantified using color deconvolution and Image-based Tool for Counting Nuclei (ITCN) plug-ins of ImageJ v1.46 software as per protocol described earlier ,,  and six mice in each group were used for quantification. Average number of Ki67 positive nuclei per animal is presented graphically and a representative image from each study group is presented in the results. "
ABSTRACT: Tissue consequences of radiation exposure are dependent on radiation quality and high linear energy transfer (high-LET) radiation, such as heavy ions in space is known to deposit higher energy in tissues and cause greater damage than low-LET γ radiation. While radiation exposure has been linked to intestinal pathologies, there are very few studies on long-term effects of radiation, fewer involved a therapeutically relevant γ radiation dose, and none explored persistent tissue metabolomic alterations after heavy ion space radiation exposure. Using a metabolomics approach, we report long-term metabolomic markers of radiation injury and perturbation of signaling pathways linked to metabolic alterations in mice after heavy ion or γ radiation exposure. Intestinal tissues (C57BL/6J, female, 6 to 8 wks) were analyzed using ultra performance liquid chromatography coupled with electrospray quadrupole time-of-flight mass spectrometry (UPLC-QToF-MS) two months after 2 Gy γ radiation and results were compared to an equitoxic (56)Fe (1.6 Gy) radiation dose. The biological relevance of the metabolites was determined using Ingenuity Pathway Analysis, immunoblots, and immunohistochemistry. Metabolic profile analysis showed radiation-type-dependent spatial separation of the groups. Decreased adenine and guanosine and increased inosine and uridine suggested perturbed nucleotide metabolism. While both the radiation types affected amino acid metabolism, the (56)Fe radiation preferentially altered dipeptide metabolism. Furthermore, (56)Fe radiation caused upregulation of 'prostanoid biosynthesis' and 'eicosanoid signaling', which are interlinked events related to cellular inflammation and have implications for nutrient absorption and inflammatory bowel disease during space missions and after radiotherapy. In conclusion, our data showed for the first time that metabolomics can not only be used to distinguish between heavy ion and γ radiation exposures, but also as a radiation-risk assessment tool for intestinal pathologies through identification of biomarkers persisting long after exposure.PLoS ONE 01/2014; 9(1):e87079. DOI:10.1371/journal.pone.0087079 · 3.23 Impact Factor
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- "A low frequency of renal Pkd1 gene inactivation and only a few renal cysts and more frequent hepatic cysts is reported from the conditional deletion of Pkd1 in MMTV-Cre mice , whereas the broadly expressed tamoxifen-Cre inducible inactivation of the Pkd1 gene in mice resulted in massive cystic transformation of renal tissue . The selective deletion of Pkd1 in kidney by using Ksp-Cre, or more broadly Nestin-Cre, also leads to the formation of polycystic kidneys resembling human ADPKD , . "
ABSTRACT: Conditional deletion of Pkd1 in osteoblasts using either Osteocalcin(Oc)-Cre or Dmp1-Cre results in defective osteoblast-mediated postnatal bone formation and osteopenia. Pkd1 is also expressed in undifferentiated mesenchyme that gives rise to the osteoblast lineage. To examine the effects of Pkd1 on prenatal osteoblast development, we crossed Pkd1(flox/flox) and Col1a1(3.6)-Cre mice, which has been used to achieve selective inactivation of Pkd1 earlier in the osteoblast lineage. Control Pkd1(flox/flox) and Pkd1(flox/+), heterozygous Col1a1(3.6)-Cre;Pkd1(flox/+) and Pkd1(flox/null), and homozygous Col1a1(3.6)-Cre;Pkd1(flox/flox) and Col1a1(3.6)-Cre;Pkd1(flox/null) mice were analyzed at ages ranging from E14.5 to 8-weeks-old. Newborn Col1a1(3.6)-Cre;Pkd1(flox/null) mice exhibited defective skeletogenesis in association with a greater reduction in Pkd1 expression in bone. Conditional Col1a1(3.6)-Cre;Pkd1(flox/+) and Col1a1(3.6)-Cre;Pkd1(flox/flox) mice displayed a gene dose-dependent decrease in bone formation and increase in marrow fat at 6 weeks of age. Bone marrow stromal cell and primary osteoblast cultures from homozygous Col1a1(3.6)-Cre;Pkd1(flox/flox) mice showed increased proliferation, impaired osteoblast development and enhanced adipogenesis ex vivo. Unexpectedly, we found evidence for Col1a1(3.6)-Cre mediated deletion of Pkd1 in extraskeletal tissues in Col1a1(3.6)-Cre;Pkd1(flox/flox) mice. Deletion of Pkd1 in mesenchymal precursors resulted in pancreatic and renal, but not hepatic, cyst formation. The non-lethality of Col1a1(3.6)-Cre;Pkd1(flox/flox) mice establishes a new model to study abnormalities in bone development and cyst formation in pancreas and kidney caused by Pkd1 gene inactivation.PLoS ONE 09/2012; 7(9):e46038. DOI:10.1371/journal.pone.0046038 · 3.23 Impact Factor
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- "Flow cytometry data were analyzed by WinMDI v2.9 and average percent change of mean fluorescence from triplicate samples of 6 mice are plotted graphically and a representative histogram comparing untreated control to treated samples in one animal is shown in the results. Images acquired from 8-oxo-dG, p-H3, and TUNEL staining were analyzed using color deconvolution and Image-based Tool for Counting Nuclei (ITCN) plug-ins of ImageJ v1.45 software by two observers blinded to treatment groups as per protocol described earlier , . We used 24 random image frames (4 frames per section; n = 6 per group) captured at 20× microscopic magnification for analysis and mean data from one experiment are presented graphically as average number of positive nuclei per 20× field and a representative image (20× magnification) from one animal of each group of one experiment is shown in the results. "
ABSTRACT: Ionizing radiation-induced oxidative stress is attributed to generation of reactive oxygen species (ROS) due to radiolysis of water molecules and is short lived. Persistent oxidative stress has also been observed after radiation exposure and is implicated in the late effects of radiation. The goal of this study was to determine if long-term oxidative stress in freshly isolated mouse intestinal epithelial cells (IEC) is dependent on radiation quality at a dose relevant to fractionated radiotherapy. Mice (C57BL/6J; 6 to 8 weeks; female) were irradiated with 2 Gy of γ-rays, a low-linear energy transfer (LET) radiation, and intestinal tissues and IEC were collected 1 year after radiation exposure. Intracellular ROS, mitochondrial function, and antioxidant activity in IEC were studied by flow cytometry and biochemical assays. Oxidative DNA damage, cell death, and mitogenic activity in IEC were assessed by immunohistochemistry. Effects of γ radiation were compared to 56Fe radiation (iso-toxic dose: 1.6 Gy; energy: 1000 MeV/nucleon; LET: 148 keV/μm), we used as representative of high-LET radiation, since it’s one of the important sources of high Z and high energy (HZE) radiation in cosmic rays. Radiation quality affected the level of persistent oxidative stress with higher elevation of intracellular ROS and mitochondrial superoxide in high-LET 56Fe radiation compared to unirradiated controls and γ radiation. NADPH oxidase activity, mitochondrial membrane damage, and loss of mitochondrial membrane potential were greater in 56Fe-irradiated mice. Compared to γ radiation oxidative DNA damage was higher, cell death ratio was unchanged, and mitotic activity was increased after 56Fe radiation. Taken together our results indicate that long-term functional dysregulation of mitochondria and increased NADPH oxidase activity are major contributing factors towards heavy ion radiation-induced persistent oxidative stress in IEC with potential for neoplastic transformation.PLoS ONE 08/2012; 7(8). DOI:10.1371/journal.pone.0042224 · 3.23 Impact Factor