Mechanoregulation of intracellular Ca2+ in human autosomal recessive polycystic kidney disease cyst-lining renal epithelial cells.
Mutations of cilia-expressed proteins are associated with an attenuated shear-induced increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in renal epithelial cell lines derived from murine models of autosomal recessive polycystic kidney disease (ARPKD). We hypothesized that human ARPKD cyst-lining renal epithelial cells also exhibited dysregulated mechanosensation. To test this, conditionally immortalized cell lines derived from human fetal ARPKD cyst-lining (pool and clone 5E) cell lines with low levels of fibrocystin/polyductin expression and age-matched normal collecting tubule [human fetal collecting tubule (HFCT) pool and clone 2C] cell lines were grown in culture, loaded with a Ca(2+) indicator dye, and subjected to laminar shear. Clonal cell lines were derived from single cells present in pools of cells from cyst-lining and collecting tubules, microdissected from human kidney. Resting and peak [Ca(2+)](i) were similar between ARPKD 5E and pool, and HFCT 2C and pool; however, the flow-induced peak [Ca(2+)](i) was greater in ARPKD 5E (700 +/- 87 nM, n = 21) than in HFCT 2C (315 +/- 58 nM, n = 12; P < 0.01) cells. ARPKD 5E cells treated with Gd(3+), an inhibitor of nonselective cation channels, inhibited but did not abolish the shear-induced [Ca(2+)](i) transient. Cilia were approximately 20% shorter in ARPKD than HFCT cells, but no difference in ciliary localization or total cellular expression of polycystin-2, a mechanosenory Gd(3+)-sensitive cation channel, was detected between ARPKD and HFCT cells. The intracellular Ca(2+) stores were similar between cells. In summary, human ARPKD cells exhibit an exaggerated Gd(3+)-sensitive mechano-induced Ca(2+) response compared with controls; whether this represents dysregulated polycystin-2 activity in ARPKD cells remains to be explored.
Rohatgi
Rajeev
R
The Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1243, New York, New York 10029, USA. Rajeev.Rohatgi@mssm.edu
Battini
Lorenzo
L
Kim
Paul
P
Israeli
Sharon
S
Wilson
Patricia D
PD
Gusella
G Luca
GL
Satlin
Lisa M
LM
D002118:Calcium / Q000502:physiology*; D018929:Cell Culture Techniques; D002923:Cilia / Q000502:physiology / Q000648:ultrastructure; D006706:Homeostasis; D006801:Humans; D007668:Kidney / Q000473:pathology / Q000503:physiopathology* / Q000601:surgery; D007685:Kidney Tubules, Collecting / Q000502:physiology / Q000503:physiopathology; D016891:Polycystic Kidney, Autosomal Dominant / Q000235:genetics / Q000473:pathology / Q000503:physiopathology* / Q000601:surgery; D011956:Receptors, Cell Surface / Q000032:analysis / Q000235:genetics; D050396:TRPP Cation Channels / Q000032:analysis; D019459:Urothelium / Q000473:pathology / Q000503:physiopathology*
D002118:Calcium; D011956:Receptors, Cell Surface; D050396:TRPP Cation Channels; C451750:PKHD1 protein, human