Major manifestations of diabetic nephropathy are increased enal hypertrophy involving glomeruli and tubules with expansion of matrix proteins, including fibronectin. These changes occur concomitant with increased expression of transforming growth factor-??(TGF?) that contributes to the pathogenesis of human and experimental diabetic nephropathy. The mechanisms by which hyperglycemia and/or TGF? ?result in hypertrophy and increased expression of fibronectin are poorly understood. We have shown recently that high glucose- as well as TGF?-induced downregulation of tumor suppressor protein PTEN (phosphatase and tensin homolog deleted on chromosome ten) contributes to renal cell hypetrophy and fibronectin expression. Our data show markedly reduced levels of PTEN in the kidney cortex and glomerulus of rats and mice with diabetes. Moreover, we demonstrate that high glucose and TGF?? increase two microRNAs, miR-21 and miR-214 in mesangial and proximal tubular epithelial cells. These microRNAs target PTEN for translational repression. In this proposal, using cultured mesangial and proximal tubular epithelial cells and renal tissues from streptozotocin-induced diabetic rats and diabetic OVE26 mice, we will test the hypothesis that transcriptional and post-transcriptional mechanisms suppress PTEN to induce hypertrophy and matrix expansion in diabetic nephropathy. Furthermore, our preliminary data demonstrate that high glucose and TGF? ?increase the levels of two transcription factors Brf1 and TBP, which cooperate with RNA polymerase III to induce 5S rRNA and tRNAs. We show an increase in the c-Myc protoonco-protein in mesangial and proximal tubular epithelial cells and in renal cortex of diabetic rats and mice. We hypothesize that the crosstalk between c- Myc and Brf1/TBP regulates diabetic renal hypertrophy and matrix protein levels. In the first specific aim, we plan to investigate the p53 tumor suppressor transcription factor as a candidate that regulates PTEN expression, hypertrophy and fibronectin expression. SIRT1 (silent information regulator 1), which deacetylates p53 to inactivate its transcriptional activity, is induced by high glucose and TGF?. We will elucidate the role of SIRT1 in cellular hypertrophy and fibronectin expression in mesangial and proximal tubular epithelial cells and in kidneys of rats and mice with diabetes. In the second aim, we will examine the role of two recently identified microRNAs, miR-21 and miR-214, in hypertrophy and fibronectin expression in response to high glucose and TGF?. In the specific aim 3, we will study the contribution of Brf1 and TBP in cooperation with c-Myc to high glucose- and TGF?-induced RNA polymerase III in regulating hypertrophy and fibronectin expression in mesangial and proximal tubular epithelial cells and in renal tissues of diabetic rats and mice. To address these specific aims, techniques including immunoprecipitation, immunoblotting, morphometry, immunohistochemistry, reporter transfection assays, chromatin immunoprecipitation assays, siRNA-mediated downregulation and conditional expression of proteins will be used.

Public Health Relevance

Manifestation of diabetic nephropathy involves increased renal ypertrophy and matrix protein amassing, which result from hyperglycemia-induced expression of transforming growth factor-??(TGF?) that along with high glucose concentration regulates expression/activation of signaling molecules. The experiments proposed in this application will identify signaling molecules, which take part in mediating renal hypertrophy and matrix protein expression. The results obtained from these experiments will help designing drugs targeting fibrosis, which constitutes the pathology of diabetic nephropathy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK050190-13
Application #
8464062
Study Section
Special Emphasis Panel (ZRG1-DKUS-G (03))
Program Officer
Flessner, Michael Francis
Project Start
1996-06-10
Project End
2014-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
13
Fiscal Year
2013
Total Cost
$294,379
Indirect Cost
$96,144
Name
University of Texas Health Science Center San Antonio
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
800772162
City
San Antonio
State
TX
Country
United States
Zip Code
78229
Das, Falguni; Bera, Amit; Ghosh-Choudhury, Nandini et al. (2014) TGF?-induced deptor suppression recruits mTORC1 and not mTORC2 to enhance collagen I (?2) gene expression. PLoS One 9:e109608
Bera, Amit; Das, Falguni; Ghosh-Choudhury, Nandini et al. (2014) microRNA-21-induced dissociation of PDCD4 from rictor contributes to Akt-IKK?-mTORC1 axis to regulate renal cancer cell invasion. Exp Cell Res 328:99-117
Mariappan, Meenalakshmi M; DeSilva, Kristin; Sorice, Gian Pio et al. (2014) Combined acute hyperglycemic and hyperinsulinemic clamp induced profibrotic and proinflammatory responses in the kidney. Am J Physiol Cell Physiol 306:C202-11
Bera, Amit; Das, Falguni; Ghosh-Choudhury, Nandini et al. (2014) A positive feedback loop involving Erk5 and Akt turns on mesangial cell proliferation in response to PDGF. Am J Physiol Cell Physiol 306:C1089-100
Lee, Doug-Yoon; Wauquier, Fabien; Eid, Assaad A et al. (2013) Nox4 NADPH oxidase mediates peroxynitrite-dependent uncoupling of endothelial nitric-oxide synthase and fibronectin expression in response to angiotensin II: role of mitochondrial reactive oxygen species. J Biol Chem 288:28668-86
Das, Falguni; Ghosh-Choudhury, Nandini; Bera, Amit et al. (2013) TGFýý-induced PI 3 kinase-dependent Mnk-1 activation is necessary for Ser-209 phosphorylation of eIF4E and mesangial cell hypertrophy. J Cell Physiol 228:1617-26
Das, Falguni; Ghosh-Choudhury, Nandini; Bera, Amit et al. (2013) Transforming growth factor ýý integrates Smad 3 to mechanistic target of rapamycin complexes to arrest deptor abundance for glomerular mesangial cell hypertrophy. J Biol Chem 288:7756-68
Ghosh-Choudhury, Nandini; Mandal, Chandi C; Das, Falguni et al. (2013) c-Abl-dependent molecular circuitry involving Smad5 and phosphatidylinositol 3-kinase regulates bone morphogenetic protein-2-induced osteogenesis. J Biol Chem 288:24503-17
Bera, Amit; Ghosh-Choudhury, Nandini; Dey, Nirmalya et al. (2013) NF?B-mediated cyclin D1 expression by microRNA-21 influences renal cancer cell proliferation. Cell Signal 25:2575-86
Lee, Hak Joo; Mariappan, Meenalakshmi M; Feliers, Denis et al. (2012) Hydrogen sulfide inhibits high glucose-induced matrix protein synthesis by activating AMP-activated protein kinase in renal epithelial cells. J Biol Chem 287:4451-61

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