, Poppas D. iSTAT system. Proliferation was also assessed in? vivo after 24?h of ureteral obstruction. There was a significant increase in HKC\8 cell number after 48?h of exposure to either 60 or 90?mmHg pressure. Western blot and qPCR confirmed increased expression of PCNA and Skp2 in pressurized cells. Cell cycle measurements demonstrated an increase in HKC\8 in S phase. Mechanical stretching increased PCNA protein expression in HKC\8 cells after 48?h while no Bufalin effect was observed on Skp2 and cell cycle measurements. Increased PCNA expression was found at 24?h after ureteral obstruction. We demonstrate direct transduction of pressure into a proliferative response in HKC\8 and NRK\52E cells, measured by cell number, PCNA and Skp2 expression and increase in cells in S phase, whereas stretch had a less robust effect on proliferation. and fibronectin in proximal tubular epithelial cells (Hamzeh et?al. 2015) and fibroblasts (El Chaar et?al. 2005; Oestergaard et?al. 2014). The obstructed kidney is usually characterized by changes in cellular proliferation, as measured by proliferating cell nuclear antigen [PCNA] and apoptosis. Truong et?al. (1996) exhibited that there was a rapid rise in proliferation Bufalin of renal tubular cells within the first 10?days of UUO; this was Bufalin followed by a decline in tubular proliferation, and an increase in tubular apoptosis. The stimulus for this is not known, but may involve mechanotransduction of the pressure signal. Cell proliferation involves progression of cells through the cell cycle; a complex network of cyclins, cyclin\dependent kinases (CDK) and CDK inhibitors [CDKI] control this process Rabbit polyclonal to EPM2AIP1 (Suzuki et?al. 2013). P27 is usually a CDKI which undergoes ubiquitination prior to its degradation, and the F\box protein Skp2 is usually a rate\limiting component of this process (Carrano et?al. 1999). Furthermore, deletion of Skp2 in the UUO model ameliorates damage, suggesting an important Bufalin role for Skp2 in the kidney (Suzuki et?al. 2007). There have been several reports of the effect of pressure and or mechanical stretch on proliferation of various cell types. Vascular easy muscle cells and bladder easy muscle cells subjected to a range of pressures in?vitro show increased proliferation (Chen et?al. 2012; Luo et?al. 2010). Gastric epithelial cells under pressure, and pulmonary epithelial cells (Chess et?al. 2005) and fibroblasts exposed to stretch also have been shown to exhibit (Nakamizo et?al. 2012; Wang et?al. 2005) increased proliferation. Interestingly, an in?vivo study of the obstructed kidney, specifically examining the urothelium covering the papilla, noted increased proliferation within 2?days of obstruction, which decreased when the obstruction was removed (Girshovich et?al. 2012). Therefore, in this study, we were interested in whether pressure or stretch would affect proliferation of human renal epithelial cells directly. Since we use the rat model of UUO extensively, we were also interested in whether pressure would also affect proliferation of rat epithelial cells. In addition to proliferation [assessed by cell counting and PCNA expression], we examined the effects of pressure and stretch on changes in cell cycle and Skp2 expression. Finally, we examined PCNA expression in?vivo at 24?h following ureteral obstruction. Materials and Methods Application of pressure to cells HKC\8 [Human renal epithelial] or NRK52E [normal rat kidney; NRK] cells were grown in a humidified atmosphere of 5% CO2\95% air at 37C in DMEM with low glucose (Gibco, Thermo Fisher Scientific, NY) supplemented with 10% FBS, penicillin and streptomycin. Cells were suspended in complete medium and cultured in 24\well plates. When cells reached 70C80% confluence, medium was changed, and in half the wells, medium was replaced with fresh serum\free medium (DMEM medium supplemented with penicillin and streptomycin only); the other half with fresh complete medium (DMEM supplemented with penicillin and streptomycin as well as 10% FBS). Pressure was applied to the cell lines through a customized pressure system developed in our laboratory (Broadbelt et?al. 2009, 2007). The cells were treated with 0, 60, or 90?mmHg pressure for 24 or 48?h. Application of stretch to cells using FlexCell apparatus The effect of stretch on HKC\8 and NRK\52E cells was studied using the FlexCell FX\5000T? system (Dunn Labortechnik GmbH, Asbach, Germany), which applies stretch to adhesive cell types.