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, 2010). The use of iCreERT2 allowed us to control expression of floxed cA-ROCK1 in the presence of tamoxifen in a temporal-specific manner in podocytes. We generated CCI-779 chemical structure mice that were heterozygous for the floxed cA-ROCK1 and carried the podocin Cre transgene (TgcA-ROCK1flox/+;Tgpodocin-iCre/+) and then activated podocin Cre by administrating tamoxifen to bitransgenic mice at a dose of 2?mg/day/mouse for a total of ten injections by 8?weeks of age ( Wang et?al., 2010). The Cre-activated bitransgenic mice (hereafter referred to as podocin Cre cA-ROCK1) were indistinguishable in size from their control littermate mice (TgcA-ROCK1flox/+ or Tgpodocin-iCre/+, hereafter referred to as control mice) ( Figure?3C). We assessed ROCK1 activation in the glomeruli of 15-week-old mice by isolating glomeruli from podocin Cre cA-ROCK1 mice 5?weeks after induction with tamoxifen ( Figures S1A�CS1C). ROCK1 activation assay revealed a ?3.5-fold increase this website of ROCK1 activation in the kidney glomeruli ( Figure?3D), whereas it was unchanged in other tissues ( Figure?S1D). Analysis of the inducible podocin Cre cA-ROCK1 mice showed significant increase in albuminuria (from 29?�� 2 to 77?�� 9 and 75?�� 6?��g/mg, p?OPHN1 mice exhibited increased mesangial matrix accumulation ( Figure?3G). However, enhanced mesangial matrix was not observed consistently in all glomeruli. The main histological change was effacement of foot processes in podocytes in podocin Cre cA-ROCK1 mice on electron microscopy ( Figure?3H). Furthermore, podocyte numbers were significantly reduced in podocin Cre cA-ROCK1 mice ( Figure?3I). Taken together, these data demonstrate that podocyte-specific knockin of cA-ROCK1 promotes albuminuria, podocyte effacement, and matrix expansion. We next explored the precise mechanisms for ROCK1-induced albuminuria. ROCK1 activation can induce apoptosis, and growing evidence has shown that mitochondrial ROS (mtROS) and apoptosis are critical in the pathogenesis of microvascular complications of diabetes, including DN (Coughlan et?al., 2007, de Cavanagh et?al., 2008, Kiritoshi et?al., 2003?and?Nishikawa et?al., 2000). Therefore, we tested the effect of ROCK1 on mtROS production in the glomeruli of control and STZ-induced diabetic mice. To specifically assess mitochondrial superoxide production, we performed Electron Paramagnetic Resonance (EPR) spectroscopy and used 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine (CMH) as the spin trap (Piskernik et?al., 2008?and?Pospisilik et?al., 2007).