Flow-Induced [Ca
2
]
i
Increase Depends on Nucleotide
Release and Subsequent Purinergic Signaling in the
Intact Nephron
Mikkel Erik Juul Jensen, Elvin Odgaard, Mette Høgh Christensen, Helle A. Praetorius, and
Jens Leipziger
Institute of Physiology and Biophysics, The Water and Salt Research Center, Aarhus University, Aarhus C, Denmark
ABSTRACT
Flow induces cytosolic Ca
2+
increases ([Ca
2+
]
i
) in intact renal tubules, but the mechanism is elusive.
Mechanical stimulation in general is known to promote release of nucleotides (ATP/UTP) and trigger
auto- and paracrine activation of P2 receptors in renal epithelia. It was hypothesized that the flow-
induced [Ca
2+
]
i
response in the renal tubule involves mechanically stimulated nucleotide release. This
study investigated (1) the expression of P2 receptors in mouse medullary thick ascending limb (mTAL)
using P2Y
2
receptor knockout (KO) mice, (2) whether flow increases induce [Ca
2+
]
i
elevations in mTAL,
and (3) whether this flow response is affected in mice that are deplete of the main purinergic receptor.
[Ca
2+
]
i
was imaged in perfused mTAL with fura-2 or fluo-4. It is shown that luminal and basolateral P2Y
2
receptors are the main purinergic receptor in this segment. Moreover, the data suggest presence of
basolateral P2X receptors. Increases of tubular flow were imposed by promptly rising the inflow
pressure, which triggered a marked increase of [Ca
2+
]
i
. This [Ca
2+
]
i
response was significantly reduced
in P2Y
2
receptor KO tubules (fura-2 ratio increase WT 0.44 0.09 [n = 28] versus KO 0.16 0.04 [n =
13]). Furthermore, the flow response was greatly inhibited with luminal and basolateral scavenging of
extracellular ATP (apyrase 7.5 U/ml) or blockage of P2 receptors (suramin 300 M). The flow response
could still be elicited in the absence of extracellular Ca
2+
. These results strongly suggest that increase
of tubular flow elevates [Ca
2+
]
i
in intact renal epithelia. This flow response is caused by release of
bilateral nucleotides and subsequent activation of P2 receptors.
J Am Soc Nephrol 18: 2062–2070, 2007. doi: 10.1681/ASN.2006070700
An increase of fluid flow over the apical side of
cultured renal epithelia
1
or in intact renal tu-
bules
2
elicits an increase of cytosolic Ca
2+
([Ca
2+
]
i
). In cultured renal epithelium, this
flow-induced [Ca
2+
]
i
response seems to depend
on the presence of the primary cilium.
3
The signal
transduction pathways of this flow-stimulated
[Ca
2+
]
i
elevation, however, is poorly under-
stood, and the available data are conflicting.
1–4
One model for the flow-induced increase in
[Ca
2+
]
i
hypothesizes that application of mechan-
ical stress/bending to the primary cilium results
in conformational changes in polycystin-1 that
further interacts with polycystin-2 to allow Ca
2+
influx at the base of the primary cilium.
4
This
Ca
2+
influx then induces Ca
2+
release from in-
tracellular stores. Noteworthy, mechanical stim-
ulation of cells including epithelia in general pro-
motes release of nucleotides (ATP, UTP).
5–9
Nucleotide release occurs without apparent dam-
age of the cells and is shown to underlie traveling
waves of [Ca
2+
]
i
in cellular networks.
7,10
Also
MDCK cells, the renal cell line used to establish
the concept of the primary cilium as mech-
Received July 6, 2006. Accepted April 10, 2007.
Published online ahead of print. Publication date available at
www.jasn.org.
M.E.J.J., E.O., and M.H.C. contributed equally to this work.
Correspondence: Dr. Jens Leipziger, Institute of Physiology and
Biophysics, The Water and Salt Research Center, Aarhus Univer-
sity, Ole Worms Alle ´ 160, 8000 Aarhus C, Denmark. Phone:
+45-89-42-2826; Fax: +45-86-12-9065; E-mail: leip@fi.au.dk
Copyright © 2007 by the American Society of Nephrology
BASIC RESEARCH
www.jasn.org
2062 ISSN : 1046-6673/1807-2062 J Am Soc Nephrol 18: 2062–2070, 2007