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Loss of TIMP3 underlies diabetic nephropathy via FoxO1/STAT1 interplay.

ABSTRACT: ADAM17 and its inhibitor TIMP3 are involved in nephropathy, but their role in diabetic kidney disease (DKD) is unclear. Diabetic Timp3(-/-) mice showed increased albuminuria, increased membrane thickness and mesangial expansion. Microarray profiling uncovered a significant reduction of Foxo1 expression in diabetic Timp3(-/-) mice compared to WT, along with FoxO1 target genes involved in autophagy, while STAT1, a repressor of FoxO1 transcription, was increased. Re-expression of Timp3 in Timp3(-/-) mesangial cells rescued the expression of Foxo1 and its targets, and decreased STAT1 expression to control levels; abolishing STAT1 expression led to a rescue of FoxO1, evoking a role of STAT1 in linking Timp3 deficiency to FoxO1. Studies on kidney biopsies from patients with diabetic nephropathy confirmed a significant reduction in TIMP3, FoxO1 and FoxO1 target genes involved in autophagy compared to controls, while STAT1 expression was strongly increased. Our study suggests that loss of TIMP3 is a hallmark of DKD in human and mouse models and designates TIMP3 as a new possible therapeutic target for diabetic nephropathy.

SUBMITTER: Fiorentino L

PROVIDER: S-EPMC3598083 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Loss of TIMP3 underlies diabetic nephropathy via FoxO1/STAT1 interplay.

Fiorentino Loredana L Cavalera Michele M Menini Stefano S Marchetti Valentina V Mavilio Maria M Fabrizi Marta M Conserva Francesca F Casagrande Viviana V Menghini Rossella R Pontrelli Paola P Arisi Ivan I D'Onofrio Mara M Lauro Davide D Khokha Rama R Accili Domenico D Pugliese Giuseppe G Gesualdo Loreto L Lauro Renato R Federici Massimo M

EMBO molecular medicine 20130212 3

ADAM17 and its inhibitor TIMP3 are involved in nephropathy, but their role in diabetic kidney disease (DKD) is unclear. Diabetic Timp3(-/-) mice showed increased albuminuria, increased membrane thickness and mesangial expansion. Microarray profiling uncovered a significant reduction of Foxo1 expression in diabetic Timp3(-/-) mice compared to WT, along with FoxO1 target genes involved in autophagy, while STAT1, a repressor of FoxO1 transcription, was increased. Re-expression of Timp3 in Timp3(-/- ...[more]

PMID: 23401241

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Project description:BackgroundDiabetic nephropathy (DN), one of the major complications of diabetes, is characterized by albuminuria, glomerulosclerosis, and progressive loss of renal function. Loss of TIMP3, an Extracellular Matrix bound protein affecting both inflammation and fibrosis, is a hallmark of DN in human subjects and mouse models.MethodsThis study was designed to provide evidences that the modulation of the system involving TIMP3 and its target A Disintegrin And Metalloproteinase 17 (ADAM17), may rescue kidney pathology in diabetic mice. Mice with cell-targeted overexpression of TIMP3 in myeloid cells (MacT3), podocyte-specific ADAM17 knockout mice (∆PodA17), and DBA/2J mice, were rendered diabetic at 8 weeks of age with a low-dose streptozotocin protocol. DBA/2J mice were administered new peptides based on the human TIMP3 N-terminal domain, specifically conjugated with G3C12, a carrier peptide highly selective and efficient for transport to the kidney. Twelve weeks after Streptozotocin injections, 24-hour albuminuria was determined by ELISA, kidney morphometry was analyzed by periodic acid-shift staining, and Real Time-PCR and western blot analysis were performed on mRNA and protein extracted from kidney cortex.ResultsOur results showed that both genetic modifications and peptides treatment positively affect renal function and structure in diabetic mice, as indicated by a significant and consistent decline in albuminuria along with reduction in glomerular lesions, as indicated by reduced mesangial expansion and glomerular hypertrophy, decreased deposition of extracellular matrix in the mesangium, diminished protein expression of the NADPH oxidases 4 (NOX4), and the improvement of podocyte structural markers such as WT1, nephrin, and podocin. Moreover, the positive effects were exerted through a mechanism independent from glycemic control.ConclusionsIn diabetic mice the targeting of TIMP3 system improved kidney structure and function, representing a valid approach to develop new avenues to treat this severe complication of diabetes.

Project description:BackgroundmiR-29c has been associated with the progression of many cancers. However, the function and mechanism of miR-29c have not been well investigated in breast cancers.MethodsReal-time quantitative PCR was used to assess expression of miR-29c and DNMT3B mRNA. Western blot and immunochemistry were used to examine the expression of DNA methyltransferase 3B (DNMT3B) protein in breast cancer cells and tissues. The functional roles of miR-29c in breast cancer cells such as proliferation, migration, invasion, colony formation, and 3D growth were evaluated using MTT, transwell chambers, soft agar, and 3D Matrigel culture, respectively. In addition, the luciferase reporter assay was used to check if miR-29c binds the 3'UTR of DNMT3B. The effects of miR-29c on the DNMT3B/TIMP3/STAT1/FOXO1 pathway were also examined using Western blot and methyl-specific qPCR. The specific inhibitor of STAT1, fludarabine, was used to further check the mechanism of miR-29c function in breast cancer cells. Studies on cell functions were carried out in DNMT3B siRNA cell lines.ResultsThe expression of miR-29c was decreased with the progression of breast cancers and was closely associated with an overall survival rate of patients. Overexpression of miR-29c inhibited the proliferation, migration, invasion, colony formation, and growth in 3D Matrigel while knockdown of miR-29c promoted these processes in breast cancer cells. In addition, miR-29c was found to bind 3'UTR of DNMT3B and inhibits the expression of DNMT3B, which was elevated in breast cancers. Moreover, the protein level of TIMP3 was reduced whereas methylation of TIMP3 was increased in miR-29c knockdown cells compared to control. On the contrary, the protein level of TIMP3 was increased whereas methylation of TIMP3 was reduced in miR-29c-overexpressing cells compared to control. Knockdown of DNMT3B reduced the proliferation, migration, and invasion of breast cancer cell lines. Finally, our results showed that miR-29c exerted its function in breast cancers by regulating the TIMP3/STAT1/FOXO1 pathway.ConclusionThe results suggest that miR-29c plays a significant role in suppressing the progression of breast cancers and that miR-29c may be used as a biomarker of breast cancers.

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Loss of TIMP3 underlies diabetic nephropathy via FoxO1/STAT1 interplay.

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Loss of TIMP3 underlies diabetic nephropathy via FoxO1/STAT1 interplay.

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