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Isolation and Epithelial Co-Culture of Mouse Renal Peritubular Endothelial

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Isolation and Epithelial Co-Culture of Mouse Renal Peritubular Endothelial Zhao et al. BMC Cell Biology 2014, 15:40 http://www.biomedcentral.com/1471-2121/15/40 METHODOLOGY ARTICLE Open Access Isolation and epithelial co-culture of mouse renal peritubular endothelial cells Ye Zhao1, Hong Zhao1,2, Yun Zhang1,3, Tania Tsatralis1, Qi Cao1, Ya Wang1, Yiping Wang1, Yuan Min Wang4, Steve I Alexander4, David C Harris1 and Guoping Zheng1* Abstract Background: Endothelial-mesenchymal transition (EndoMT) has been shown to be a major source of myofibroblasts, contributing to kidney fibrosis. However, in vitro study of endothelial cells often relies on culture of isolated primary endothelial cells due to the unavailability of endothelial cell lines. Our recent study suggested that peritubular endothelial cells could contribute to kidney fibrosis through EndoMT. Therefore, successful isolation and culture of mouse peritubular endothelial cells could provide a new platform for studying kidney fibrosis. This study describes an immunomagnetic separation method for the isolation of mouse renal peritubular endothelial cells using anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain endothelial phenotype. Results: Flow cytometry showed that after isolation and two days of culture, about 95% of cells were positive for endothelial-specific marker CD146. The percentage of other cells, including dendritic cells (CD11c) and macrophages (F4/80), was less than 1%. Maintenance of endothelial cell phenotype required vascular endothelial growth factor (VEGF) and co-culture with mouse proximal tubular epithelial cells. Conclusion: In this study, we established a method for the isolation of mouse renal peritubular endothelial cells by using immunomagnetic separation with anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain phenotype. Keywords: Peritubular endothelial cells, Tubular epithelial cells, CD146, Co-culture, Vascular endothelial growth factor Background of EndoMT-derived fibroblasts. However, elucidation of Endothelial cells have been found to be a major source molecular mechanisms of EndoMT involving glomerular of myofibroblasts via endothelial mesenchymal transition or peritubular endothelial cells relies largely on in vitro (EndoMT), causing fibrosis in kidney and other organs studies using primary isolated human or mouse endothe- [1-4]. In one key study, Zeisberg and colleagues [4] lial cells. Such studies are limited by the loss of phenotype showed in three mouse models including unilateral ur- that occurs in those primary endothelial cells in culture eteral obstructive nephropathy (UUO), Alport disease and after a limited number of passages. streptozotocin-induced diabetic nephropathy, that around Renal endothelial cells include glomerular endothelial 30% to 50% of fibroblasts formed in the kidneys co- cells, peritubular endothelial cells and vascular endothe- expressed the endothelial marker CD31 and the fibroblast/ lial cells. Although it is generally accepted that endothe- myofibroblast markers fibroblast specific protein-1 (FSP-1) lial cells contribute to fibroblast formation in kidney, the and/or α-smooth muscle actin (α-SMA). Endothelial contribution of different renal endothelial cells has not lineage tracing using Tie2-Cre;R26R-stop-EYFP transgenic been defined. Previous studies examining EndoMT in mice in the UUO model [4] and in streptozotocin-induced renal fibrosis were mostly focused on glomerular endothe- diabetic nephropathy [5] further confirmed the presence lial cells, not surprisingly using the well-established method for isolation of glomerular endothelial cells [6-9]. * Correspondence: [email protected] By immunofluorescence staining of kidney sections of 1Centre for Transplant and Renal Research, Westmead Millennium Institute, The University of Sydney, Sydney, NSW, Australia mice with UUO, co-localization of the mesenchymal Full list of author information is available at the end of the article marker α-SMA and endothelial marker CD31 or VE- © 2014 Zhao et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhao et al. BMC Cell Biology 2014, 15:40 Page 2 of 10 http://www.biomedcentral.com/1471-2121/15/40 cadherin was observed predominantly outside glomeruli, solution (Table 1) and incubated at 37°C in a gentle shak- suggesting that the interstitial peritubular rather than ing water bath for 15 min. The suspension was homoge- glomerular endothelial cells play the major role, at least in nized by pipetting 5 to 10 times through a sterile transfer the UUO model. To date, however, a method for isolation pipette followed by addition of 1 ml of fresh collagenase of peritubular endothelial cells of high purity has not been type IV solution. This process was repeated 2-4 times. described [10]. For example, the method described by About 40 ml fresh ice-cold DMEM/F12 was then added Mcginn et al. [8] may isolate lymphatic and vascular into the collagenase digestion solution and the suspension endothelial cells. was centrifuged at 200 × g for 2 min. The pellet was resus- Primary endothelial cells are susceptible to phenotypic pended and washed in 10 ml of fresh ice-cold DMEM/F12 change in culture; a co-culture system was, therefore, and centrifuged at 150 × g for 2 min at 4°C. Density- developed to mimic the in vivo micro-environment in gradient centrifugation of the pellet was then performed the kidney with its key interactions between renal tubu- by resuspension in 25 ml of 45% (vol/vol) sterile Percoll lar epithelial cells and adjacent endothelial cells. Tasnim solution (Table 2) in 50 ml centrifugation tubes and et al. [10] described interactions by which human renal centrifugation at 5525 × g for 30 min at 4°C (without glomerular endothelial cells improved the stability of the braking). After centrifugation, the tubule fractions were human renal tubular cell phenotype while glomerular collected from the top layer of the Percoll solution (5 ml endothelial cell phenotype was also well-maintained by of the top layer). The tubule fraction was washed once in tubular epithelial cells. However, such a system may not 20 ml ice-cold DMEM/F12 medium at 300 × g for 5 min be applicable to the interaction between peritubular at 4°C and resuspended for further experiments. endothelial cells and tubular epithelial cells in vivo. Here we describe a method for isolation of primary Isolation of proximal tubular epithelial cells from tubule mouse renal peritubular endothelial cells (MRPEC) and fraction co-culture with mouse renal proximal tubular epithelial The pellet was resuspended in K1 medium [DMEM: cells. Our results show that separation with anti-CD146 HAM’s F12; 1:1 vol/vol; Gibco Life Technologies) sup- MicroBeads resulted in peritubular endothelial cells of plemented with 25 μg/ml of epithelial growth factor high purity. Further, co-culture with mouse renal pro- (EGF; Sigma Chemical Co.; St. Louis, MO, USA), 25 μM ximal tubular epithelial cells maintained the phenotype HEPES (Invitrogen; Carlsbad, CA, USA), hormone mix- of isolated peritubular endothelial cells, thus providing a ture (Table 3) and 5% fetal calf serum (FCS; Invitrogen)]. stable in vitro model for investigating the role of peri- Proximal tubular epithelial cells were obtained by direct tubular endothelial cells in kidney diseases. culture after removing peritubular endothelial cells. Methods Isolation of peritubular endothelial cells from tubule Animals fraction Male BALB/c mice (6 week old) were purchased from The tubule fractions were further digested with 0.025% Australian Research Council and experiments were trypsin or collagenase type IV solution for 10 to 15 min performed in accordance with protocols approved by at 37°C to obtain single cell suspensions. After digestion, Animal Ethics Committee of Western Sydney Local ice-cold PBS was added to the cell suspension followed Health District. by filtering sequentially through a 70-μm and a 40-μm cell strainer. The filtered cell suspensions were then cen- Separation of tubular fraction from kidney cortex trifuged at 300 × g for 2 min at 4°C. The pellet was re- Mouse kidney tubular fractions were obtained from the suspended in MicroBeads resuspension buffer [prepared kidney cortex of BALB/c mice using established methods in PBS, containing 0.5% BSA (Sigma Chemical Co.) and adapted from Doctor et al. [11]. Kidneys were perfused 2 mM EDTA (Life Technologies)] and centrifuged again in situ via the aorta with 20 ml phosphate buffered saline at 300 × g for 3 min at 4°C. The cell pellets obtained (PBS; Lonza; Walkersville, MD, USA) containing 80U/ml heparin to remove blood from anesthetized mice. Kidney Table 1 Components of collagenase type IV solution capsule was removed by peeling with forceps. Freshly iso- Collagenase D solution Volume or Supplier concentration lated kidneys were placed in ice-cold Dulbecco’sModified ’ ’ DMEM/F12 medium 7.5 ml Gibco Life Technologies; Eagles Medium mixed with Hams F12 (DMEM/F12; 1:1 Grand Island, NY, USA ratio; Gibco Life Technologies; Grand Island, NY, USA) Collagenase type IV 1 mg/ml Sigma Chemical Co.; on a petri dish. The kidney was sliced coronally and St Louis, MO, USA homogenized by mincing into 1 mm3 to 2 mm3 pieces. Deoxyribonuclease 0.1 mg/ml Sigma Chemical Co. The homogenized kidney cortex tissue pieces were re- Bovine serum albumin (BSA) 1 mg/ml Sigma Chemical Co. suspended and mixed in 7.5 ml of collagenase type IV Zhao et al. BMC Cell Biology 2014, 15:40 Page 3 of 10 http://www.biomedcentral.com/1471-2121/15/40 Table 2 Percoll solution (25 ml) cells were then washed twice in cold PBS with 1% FCS Percoll solution Volume Supplier and analyzed using a flow cytometer (BD Bioscience; DMEM/F12 medium 12.5 ml Gibco Life Technologies; San Jose, CA, USA).
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