TY - JOUR
T1 - Characterization of a long-term rat mTAL cell line
AU - Eng, Ben
AU - Mukhopadhyay, Somshuvra
AU - Vio, Carlos P.
AU - Pedraza, Paulina L.
AU - Hao, Shoujin
AU - Battula, Sailaja
AU - Sehgal, Pravin B.
AU - McGiff, John C.
AU - Ferreri, Nicholas R.
PY - 2007/10
Y1 - 2007/10
N2 - A medullary thick ascending limb (mTAL) cell line, termed raTAL, has been established from freshly isolated rat mTAL tubules and cultured continuously for up to 75 passages; it retains characteristics of mTAL cells even after retrieval from storage in liquid nitrogen for several months. The cells express Tamm-Horsfall glycoprotein (THP), a TAL-specific marker, grow to confluence, exhibit a polygonal morphology characteristic of epithelial cells, and form "domes." Detection of THP, Na+-K+-2Cl - cotransporter (NKCC2), Na+-K+-ATPase, and renal outer medullary K+ channel (ROMK) was achieved using indirect immunofluorescence and confocal microscopy. Western blot analysis of NKCC2 expression using two different antibodies revealed a band of ∼160 kDa, and RT-PCR analysis demonstrated the presence of NKCC2 isoforms A and F, which was confirmed by DNA sequencing; transport of Cl- into raTAL cells was inhibited by furosemide. Ouabainand bumetanide-sensitive oxygen consumption, an index of ion transport activity in the mTAL, was observed in raTAL cells, and the number of domes present was reduced significantly when cells were incubated in the presence of ouabain or bumetanide. The specific activity of Na +-K+-ATPase activity was determined in raTAL cells (0.67 ± 0.18 nmol Pi·μg protein -1·min-1), primary cultures of mTAL cells (0.39 ± 0.08 nmol Pi·μg protein -1·min-1), and freshly isolated mTAL tubules (1.10 ± 0.29 nmol Pi·μg protein -1·min-1), and ∼30-50% of total cellular ATPase activity was inhibited by ouabain, in accord with other mTAL preparations. This cell line will be used in studies that address biochemical, molecular, and physiological mechanisms in the mTAL.
AB - A medullary thick ascending limb (mTAL) cell line, termed raTAL, has been established from freshly isolated rat mTAL tubules and cultured continuously for up to 75 passages; it retains characteristics of mTAL cells even after retrieval from storage in liquid nitrogen for several months. The cells express Tamm-Horsfall glycoprotein (THP), a TAL-specific marker, grow to confluence, exhibit a polygonal morphology characteristic of epithelial cells, and form "domes." Detection of THP, Na+-K+-2Cl - cotransporter (NKCC2), Na+-K+-ATPase, and renal outer medullary K+ channel (ROMK) was achieved using indirect immunofluorescence and confocal microscopy. Western blot analysis of NKCC2 expression using two different antibodies revealed a band of ∼160 kDa, and RT-PCR analysis demonstrated the presence of NKCC2 isoforms A and F, which was confirmed by DNA sequencing; transport of Cl- into raTAL cells was inhibited by furosemide. Ouabainand bumetanide-sensitive oxygen consumption, an index of ion transport activity in the mTAL, was observed in raTAL cells, and the number of domes present was reduced significantly when cells were incubated in the presence of ouabain or bumetanide. The specific activity of Na +-K+-ATPase activity was determined in raTAL cells (0.67 ± 0.18 nmol Pi·μg protein -1·min-1), primary cultures of mTAL cells (0.39 ± 0.08 nmol Pi·μg protein -1·min-1), and freshly isolated mTAL tubules (1.10 ± 0.29 nmol Pi·μg protein -1·min-1), and ∼30-50% of total cellular ATPase activity was inhibited by ouabain, in accord with other mTAL preparations. This cell line will be used in studies that address biochemical, molecular, and physiological mechanisms in the mTAL.
KW - Cell culture
KW - Medullary thick ascending limb
KW - Renal cell lines
KW - Renal epithelial cells
UR - http://www.scopus.com/inward/record.url?scp=35348883634&partnerID=8YFLogxK
U2 - 10.1152/ajprenal.00426.2006
DO - 10.1152/ajprenal.00426.2006
M3 - Article
C2 - 17670898
AN - SCOPUS:35348883634
SN - 1931-857X
VL - 293
SP - F1413-F1422
JO - American Journal of Physiology - Renal Physiology
JF - American Journal of Physiology - Renal Physiology
IS - 4
ER -