Establishment of conditionally immortalised human glomerular mesangial

Ramadan M Sarrab, Rachel Lennon, Lan Ni, Matthew D Wherlock, Gavin I Welsh, Moin 3 A Saleem 4 Children’s Renal Unit and Academic Renal Unit, University of Bristol, Learning and Research Building, 5 Southmead Hospital, Bristol, UK; 6 7 8 Running Head: Conditionally immortalized human mesangial cells 9 10 Corresponding Author: Moin A Saleem. [email protected] 11 Academic Renal Unit, Learning and Research Building, Southmead Hospital, Bristol BS10 5NB, UK 12 13 14 Background The aim of this study was to establish an immortalized human mesangial cell line 15 similar to mesangial cells in vivo for use as a tool for understanding glomerular cell function. 16 Methods Mesangial cells were isolated from glomerular outgrowths from a normal human 17 kidney, then retrovirally transfected with a temperature sensitive SV40T antigen + human 18 telomerase (hTERT). 19 Results Mesangial cells exhibited features of compact cells with small bodies in a confluent 20 monolayer at 33°C but the cell shape changed to flat and stellate after 5 days in growth 21 restrictive conditions (37°C). Western blot and immunofluorescence analysis showed that 22 podocyte markers (nephrin, CD2AP, podocin, WT1) and an endothelial specific molecule (VE23 cadherin) were not detectable in this cell line, whereas markers characteristic of mesangial cells 24 (α-SMA, Fibronectin and PDGFβ-R were strongly expressed. In migration assays, a significant 25 reduction in wound surface was observed in podocyte and endothelial cells as soon as 12 hours 26 (75% and 62% respectively) and complete wound closure after 24 hours. In contrast, no 27 significant change was observed in mesangial cells after 12 hours and even after 48 hours the 28 wounds were not completely closed. 29 Discussion Until now conditionally immortalized podocyte and endothelial cell lines derived 30 from mouse and human have been described and this has greatly boosted research on glomerular 31 physiology and pathology. We have established the first conditionally immortalized human 32 glomerular mesangial cell line, which will be an important adjunct in studies of representative 33 glomerular cells, as well as in co-culture studies. Unexpectedly mesangial cells ability to migrate 34 Articles in PresS. Am J Physiol Renal Physiol (June 8, 2011). doi:10.1152/ajprenal.00589.2010