Original Data
| Rev Diabet Stud,
2005,
2(1):19-26 |
DOI 10.1900/RDS.2005.2.19 |
Proteomic Identification and Immunolocalization of Increased Renal Calbindin-D28k Expression in OVE26 Diabetic Mice
Visith Thongboonkerd1,2, Shirong Zheng3, Kenneth R. McLeish1,4,5, Paul N. Epstein3,6, Jon B. Klein1,4,5
1Core Proteomics Laboratory, Kidney Disease Program, Department of Medicine, University of Louisville, Louisville, KY, USA.
2Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine at Siriraj Hospital, Mahidol University, Bangkok, Thailand.
3Department of Pediatrics, University of Louisville, Louisville, KY, USA.
4Veterans Affairs Medical Center, Louisville, KY, USA.
5Department of Biochemistry and Molecular Biology, University of Louisville, Louisville, KY, USA.
6Department of Pharmacology and Toxicology, University of Louisville, Louisville, KY, USA.
Address correspondence to: Visith Thongboonkerd, e-mail: thongboonkerd@dr.com.
Abstract
Diabetic nephropathy is a common diabetic complication that is associated with alterations in the expression of several renal proteins and abnormal calcium homeostasis. We performed proteomic analysis to screen for global changes of renal protein expression in diabetic kidney. Proteins extracted from the whole kidney of 120-day-old OVE26 (a transgenic model of Type 1 diabetes) and FVB (non-diabetic background strain) mice were separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and visualized by SYPRO Ruby staining (n = 5 in each group). Quantitative intensity analysis revealed 41 differentially expressed proteins, of which 30 were identified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) followed by peptide mass fingerprinting. One of the altered proteins with the greatest magnitude of change was the calcium-binding protein, calbindin-D28k, whose expression was increased 6.7-fold in diabetic kidney. We confirmed the increase in calbindin-D28k expression in diabetic kidney by Western blot analysis. Immunohistochemical study demonstrated that calbindin-D28k expression was markedly increased in tubular epithelial cells of distal convoluted tubules (DCT), collecting ducts (CD), and proximal convoluted tubules (PCT) in diabetic kidney. Calbindin-D28k plays a critical role in maintaining calcium homeostasis. The elevation in renal calbindin-D28k expression in our model may indicate a compensatory mechanism to overcome hypercalciuria in diabetes.
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| Rev Diabet Stud,
2005,
2(1):27-34 |
DOI 10.1900/RDS.2005.2.27 |
Diabetes-Induced Fetal Growth Retardation is Associated with Suppression of NF-κB Activity in Embryos
Keren Mammon, Rotem Keshet, Shoshana Savion, Olga Pekar, Zeev Zaslavsky, Amos Fein, Vladimir Toder, Arkady Torchinsky
Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel.
Address correspondence to: Arkady Torchinsky, e-mail: arkadyt@post.tau.ac.il
Abstract
BACKGROUND: Mechanisms underlying diabetes-induced fetal growth retardation remain largely undefined. Two events such as the persistent activation of apoptosis or suppression of cell proliferation in embryos might directly result in fetal growth retardation. Evidence implicating the transcription factor NF-κB in the regulation of the physiological and teratogen-induced apoptosis as well as cell proliferation suggests that it may be a component of mechanisms underlying this pathology. To address this issue, this study was designed to test: 1) whether diabetes-induced fetal growth retardation is preceded by the modulation of NF-κB activity in embryos at the late stage of organogenesis and 2) whether apoptosis is altered in these embryos. METHODS: The embryos and placentas of streptozotocin-induced diabetic mice collected on days 13 and 15 of pregnancy were used to evaluate the expression of NF-κB, IκBα and phosphorylated (p)-IκBα proteins by Western blot analysis and NF-κB DNA binding by an ELISA-based method. The detection of apoptotic cells was performed by the TUNEL assay and the expression of a proapoptotic protein Bax was evaluated by the Western blot. RESULTS: The embryos of diabetic mice were significantly growth retarded, whereas the placental weight did not differ in diabetic or control females. Levels of NF-κB and p-IκBα proteins as well as the amount of NF-κB DNA binding was lower in embryos of diabetic mice as compared to those in controls. However, neither excessive apoptosis nor an increased Bax expression was found in growth-retarded embryos and their placentas. CONCLUSION: The study herein revealed that diabetes-induced fetal growth retardation is associated with the suppression of NF-κB activity in embryos, which seems to be realized at the level of IκB degradation.
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