Nephrology Research and Training Center, Division of Nephrology, Departments of Medicine and Physiology, University of Alabama at Birmingham, Birmingham, Alabama 35294
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ABSTRACT |
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Previous
micropuncture studies suggested that macula densa (MD) cells might
detect variations in luminal sodium chloride concentration ([NaCl]l) through
changes in cytosolic calcium
([Ca2+]c).
To test this hypothesis, MD
[Ca2+]c
was measured with fluorescence microscopy using fura 2 in the isolated
perfused thick ascending limb with attached glomerulus preparation
dissected from rabbit kidney. Tubules were bathed and perfused with a
Ringer solution,
[NaCl]l was varied and
isosmotically replaced with
N-methyl-D-glucamine
cyclamate. Control
[Ca2+]c,
during perfusion with 25 mM NaCl and 150 mM NaCl in the bath, averaged
101.6 ± 8.2 nM (n = 21).
Increasing [NaCl]l to
150 mM elevated
[Ca2+]c
by 39.1 ± 5.2 nM (n = 21, P < 0.01). This effect was
concentration dependent between zero and 60 mM
[NaCl]l. The presence
of either luminal furosemide or basolateral nifedipine or
5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a potent
Cl channel blocker,
significantly reduced resting
[Ca2+]c
and abolished the increase in
[Ca2+]c
in response to increased
[NaCl]l. Nifedipine
failed to produce a similar inhibitory effect when added exclusively to
the luminal perfusate. Also, 100 nM BAY K 8644, a voltage-gated
Ca2+ channel agonist, added to the
bathing solution increased
[Ca2+]c
by 33.2 ± 8.1 nM (n = 5, P < 0.05). These
observations suggest that MD cells may detect variations in
[NaCl]l through a
signaling pathway that includes
Na+-2Cl
-K+
cotransport, basolateral membrane depolarization via
Cl
channels, and
Ca2+ entry through voltage-gated
Ca2+ channels.
isolated perfused tubule; fluorescence microscopy; cytosolic calcium; furosemide; voltage-gated calcium channels; tubuloglomerular feedback
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INTRODUCTION |
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MACULA DENSA (MD) cells, which are located within the
cortical thick ascending limb (CTAL), detect changes in
tubular fluid sodium chloride concentration
([NaCl]l) and
transmit signals to the preglomerular vascular elements
(tubuloglomerular feedback, TGF) (1, 2, 17). The mechanism by which
these cells sense changes in tubular fluid composition has remained
elusive, but is thought to involve specific ionic transport processes.
In addition to the well-known furosemide/bumetanide-sensitive
Na+-2Cl-K+
cotransporter (6, 9, 10), the apical membrane contains a
Na+/H+
exchanger and a high density of K+
channels (6). At the basolateral membrane, NaCl exit may occur, at
least in part, through
Na+-K+-ATPase
and Cl
channels (6, 9, 11).
Previous micropuncture studies (4, 5) from our laboratory suggested that a cytosolic Ca2+ system might be involved in the MD cell signaling. In these studies, luminal perfusion of the Ca2+ ionophore, A23187, in the presence of perfusate Ca2+ enhanced feedback responses (4), whereas 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), an inhibitor of intracellular release of Ca2+, reduced stop-flow pressure feedback responses (5). An MD cytosolic calcium ([Ca2+]c) signaling pathway, involved in TGF signal transmission, was appealing, since Ca2+ could have a number of potential effects including regulation of ionic transport processes (6) or stimulation of the release of a chemical mediator. However, more recent studies (7, 12; and unpublished observations) using fluorescence microscopy and the isolated perfused tubule technique to measure MD [Ca2+]c directly in response to changes in [NaCl]l have produced equivocal results. It is therefore uncertain whether a rise in [NaCl]l does in fact result in an elevation in MD [Ca2+]c. Part of the problem has been difficulties (at least in our laboratory) in using fura 2 to measure [Ca2+]c in MD cells. Several technical modifications have recently been made that have allowed us to reliably measure changes in MD [Ca2+]c using fluorescence microscopy.
Therefore, the purpose of this study was to reassess the effects of changes in [NaCl]l on MD [Ca2+]c. Our work is the first to report that increases in [NaCl]l do indeed produce significant increases in MD [Ca2+]c. We went on to then examine the mechanism by which increased [NaCl]l induces elevations in MD [Ca2+]c.
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METHODS |
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CTAL preparation. Individual CTALs with attached glomeruli were dissected from rabbit kidney and perfused in vitro as described previously (13) with minor modifications. To prevent intracellular accumulation of NaCl prior to the experiment, the dissection solution was an isosmotic, low-NaCl Ringer solution consisting of (in mM) 25 NaCl, 120 N-methyl-D-glucamine (NMDG) cyclamate, 5 KCl, 1 MgSO4, 1.6 Na2HPO4, 0.4 NaH2PO4, 1.5 CaCl2, 5 D-glucose, and 10 HEPES. Temperature was maintained at about 4°C. An individual CTAL was transferred to a chamber mounted on an inverted microscope. The tubule was kept in the low-NaCl solution until it was cannulated and perfused with the isosmotic 25 mM NaCl solution. Then the bathing solution was changed to the 150 mM NaCl Ringer solution at 37°C. According to our experience, this maneuver greatly improved the responsiveness of MD cells, at least in terms of [Ca2+]c dynamics. When we used a Ringer dissection solution containing 150 mM NaCl, we got the same equivocal results reported by others, i.e., increases, decreases, or no changes in [Ca2+]c with alterations in luminal composition.
Measurement of [Ca2+]c. [Ca2+]c of MD cells was measured with dual-excitation wavelength fluorescence microscopy (Photon Technologies, Princeton, NJ) using the fluorescent probe fura 2 (Teflabs, Austin, TX). Fura 2 fluorescence was measured at an emission wavelength of 510 nm in response to excitation wavelengths of 340 and 380 nm, alternated at a rate of 25 Hz by a computer-controlled chopper assembly. An adjustable photometer window was positioned over the MD plaque (consisting of ~10-15 cells), and emitted photons were detected by a Leitz photometer that was modified for photon counting. Magnification was ×400 using an Olympus ×40 UVFL lens. Autofluorescence-corrected ratios (340 nm/380 nm) were calculated at a rate of 5 points/s using PTI software. MD cells were loaded with the dye by adding the acetoxymethyl ester of fura 2 (fura 2-AM, 10 µM) dissolved in DMSO to the 25 mM NaCl containing luminal perfusate. Loading required ~15 min, after which fura 2-AM was removed from the lumen. A ~20-min incubation of the tubule with the control perfusion solution was allowed to permit stabilization of fluorescent signal.
Increases in [Ca2+]c normally result in an increase in the fura 2 340-nm signal and a decrease in the 380-nm signal. In MD cells, progressing from a perfusate of 25 mM NaCl Ringer solution (
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RESULTS |
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Basal and
[NaCl]l-dependent
Ca2+
activity.
Resting MD
[Ca2+]c,
in the presence of 25 mM
[NaCl]l, was 101.6 ± 8.2 nM (n = 21). As shown in a
representative recording in Fig. 1, and
summarized in Fig. 2, increasing
[NaCl]l from 25 to 150 mM caused a rapid and reversible increase in
[Ca2+]c,
which averaged 39.1 ± 5.2 nM (n = 21, P < 0.01). Also, this [NaCl]l-induced
elevation in
[Ca2+]c
was NaCl concentration dependent between 0 and 60 mM
[NaCl]l. Normalized as
percentage of the basal value of 65.1 ± 3.8 nM in the presence of
zero [NaCl]l,
increasing [NaCl]l to
25, 40, 50 and 60 mM increased
[Ca2+]c
by 21 ± 4, 29 ± 4, 37 ± 4, and 49 ± 3%,
respectively (n = 5, P < 0.05 compared with each
preceding value). Also, as shown in Fig. 2, addition of 50 µM
furosemide to the luminal perfusate significantly reduced resting
[Ca2+]c
and abolished
[NaCl]l-induced
elevations in
[Ca2+]c.
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Mechanism of increase in [Ca2+]c. To determine the source for the increase in [Ca2+]c, we first examined the effect of TMB-8, a putative inhibitor of intracellular release of bound Ca2+. Addition of 500 µM TMB-8 to the bath had no effect on the [Ca2+]c response; increasing [NaCl]l from 25 to 150 mM increased [Ca2+]c by 35.5 ± 12.3 nM (n = 5, P < 0.05), a value not significantly different from control.
It is well established that progressive increases in [NaCl]l between 20 to 60 mM result in parallel depolarization of the MD cell basolateral membrane (3, 6). Therefore, the presence of a membrane voltage-associated Ca2+ pathway seemed plausible. To examine the role of putative voltage-gated Ca2+ channels in the MD [Ca2+]c response to increased [NaCl]l, experiments were performed utilizing the dihydropyridine Ca2+ antagonist nifedipine. As shown in Fig. 2, the increase in MD [Ca2+]c normally elicited by 150 mM [NaCl]l was abolished in the presence of 1 µM nifedipine added to the bath. Furthermore, bath nifedipine caused a significant reduction of the MD resting [Ca2+]c. In contrast, nifedipine failed to produce similar inhibitory effects when added exclusively to the luminal perfusate. Increasing [NaCl]l from 25 to 150 mM in the presence of 1 µM luminal nifedipine increased [Ca2+]c by 37.3 ± 6.2% (n = 6, P < 0.05) from a resting value of 100.3 ± 10.7 nM, an increase that was not significantly different from that found in the absence of luminal nifedipine. Basolateral Cl ![]() |
DISCUSSION |
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In the present studies, we measured [Ca2+]c in MD cells with fluorescence microscopy using the isolated perfused CTAL-MD preparation. Resting [Ca2+]c during perfusion with 25 mM NaCl and 150 mM NaCl in the bath averaged 101.6 ± 8.2 nM, a value within the range that has been reported previously (2, 7, 14). Increases in [NaCl]l from 25 to 150 mM resulted in consistent increases in MD [Ca2+]c. The reason why we have now obtained consistent MD [Ca2+]c responsiveness may be the result of two technical modifications. First was the use of a low-NaCl dissection solution, which should minimize luminal NaCl entry and prevent prolonged high intracellular [Na+] and [Ca2+]c. Second, was the fact that all solutions were isosmotic, which eliminated cell volume-related changes in fura 2 fluorescence (unpublished observations). Salomonsson et al. (7, 14) reported no consistent changes in MD [Ca2+]c; however, these experiments were performed using hypotonic luminal perfusates, which may have interfered with the detection of changes in [Ca2+]c.
[NaCl]l-induced Ca2+ responses were also found to be concentration dependent between zero and 60 mM [NaCl]l, the most sensitive range for changes in TGF responses (2, 6, 17). These findings are consistent with the conclusion of previous micropuncture experiments (4, 5) that MD cells might detect variations in tubular fluid composition and transmit vasoconstrictor feedback signals to the afferent arteriole through a cytosolic Ca2+ system.
It is generally accepted that generation of feedback signals involves,
at least in part, changes in
[NaCl]l. At the apical membrane, the primary NaCl entry site is through the
Na+-2Cl-K+
cotransporter (6, 10), whereas NaCl exit may occur, at
least in part, through
Na+-K+-ATPase
and a Cl
conductance (6, 9,
11). Secondary changes in cell
[Cl
], as the
result of alterations in NaCl transport by MD cells, acting at the
basolateral Cl
channel (9)
may be responsible for the changes in basolateral membrane potential
(Vbl) that are
observed in these cells (3, 10, 15, 16). The regulation of
Vbl by
intracellular Cl
was
established in previous microelectrode and ion substitution studies (3,
6, 10, 11). These studies indicated that apical
Na+-2Cl
-K+
cotransport by MD cells would result in an elevation in intracellular Cl
activity, electrogenic
Cl
efflux across the
basolateral membrane, and depolarization of Vbl. This general
notion has been supported by Schlatter et al. (15, 16), who showed that
addition of NPPB to the bathing solution hyperpolarized MD cells. Thus
progressive increases in [NaCl]l from 20 to 60 mM result in parallel changes in TGF responses (vasoconstriction) and
membrane depolarization (6).
Shown in Fig. 3 is a schematic drawing of
how such transport-related events may increase MD
[Ca2+]c.
In support of this model, we found that luminal furosemide abolished
elevations in MD
[Ca2+]c
in response to an increase in
[NaCl]l. From this
observation, we conclude that the apical
Na+-2Cl-K+
cotransporter is involved in the MD
Ca2+ response. In previous work
measuring MD Vbl
(3, 10), we found that progressive increases in
[NaCl]l from 20 to 60 mM resulted in progressive depolarization of the MD
Vbl and that
furosemide abolished
[NaCl]l-induced MD
depolarization. Schlatter et al. (15, 16) likewise found that
furosemide hyperpolarized MD cells.
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It was therefore reasoned that this large depolarization of Vbl, in the presence of high [NaCl]l, could serve to promote calcium entry across the basolateral membrane. We therefore examined the presence of putative voltage-gated Ca2+ channels in MD cells. Addition of the dihydropyridine Ca2+ antagonist nifedipine to the bathing solution abolished the [NaCl]l-induced increase in MD [Ca2+]c and significantly reduced resting [Ca2+]c. Also, BAY K 8644, a voltage-gated Ca2+ channel agonist, added to the bath significantly increased MD [Ca2+]c. These data strongly support the functional expression of voltage-gated Ca2+ channels at the basolateral membrane of MD cells.
Since basolateral Cl
channels are responsible for the depolarization of MD
Vbl with
increases in [NaCl]l
(6, 9, 11, 16), another test of this model was to determine the effect of inhibiting the basolateral
Cl
channels.
[NaCl]l-induced
changes in MD
[Ca2+]c
were evaluated in the presence of the
Cl
channel blocker NPPB.
Addition of NPPB to the bathing solution, significantly reduced resting
[Ca2+]c
and abolished the elevation in MD
[Ca2+]c
in response to increasing
[NaCl]l. Thus these
results further support a role for depolarization-induced
Ca2+ entry across the MD
basolateral membrane.
In most cell types, sustained activation of a cytosolic calcium system requires both Ca2+ mobilization as well as Ca2+ entry. In earlier micropuncture studies (5), a role for Ca2+ entry across the luminal membrane was tested by removal of luminal Ca2+ and addition of a Ca2+ chelator. In the presence of an isotonic solution, deletion of luminal Ca2+ did not influence feedback responses, eliminating a role for Ca2+ entry into the MD cells across the luminal membrane. However, Naruse et al. (12) found alterations in the afferent arteriolar diameter in response to changes in luminal [Ca2+]. Based on this finding, they suggested the presence of an apical dihydropyridine-sensitive Ca2+ conductance. In contrast, the present studies showed that administration of nifedipine to the luminal perfusate had no effect on either the resting [Ca2+]c or the [NaCl]l-induced Ca2+ response of MD cells. Thus our data support the conclusion of earlier micropuncture experiments, indicating the absence of apical voltage-gated Ca2+ channels in MD cells.
In previous micropuncture experiments (5), luminal administration of TMB-8, a putative inhibitor of intracellular release of bound Ca2+, resulted in a substantial reduction in the magnitude of feedback responses. This inhibition was specific for Ca2+, since inhibition by TMB-8 could be overcome by simultaneous addition of a Ca2+ ionophore. In the present studies, TMB-8 had no effect on MD [Ca2+]c dynamics, suggesting that Ca2+ entry via basolateral voltage-gated Ca2+ channels may be the primary source for the [NaCl]l-induced Ca2+ signal. However, these studies cannot entirely rule out the possibility that Ca2+ mobilization may occur in response to elevated [NaCl]l. It should be noted that previous work from our laboratory (2, 4) has reported that changes in luminal osmolality can also influence feedback responses. It is possible that this occurs through alterations in cell volume, which could have an effect on [Ca2+]c and possibly on Ca2+ mobilization. Unfortunately, due to technical constraints, it was not possible to evaluate the effects of changes in perfusate osmolality on MD [Ca2+]c. Nevertheless, the current studies support a model in which Ca2+ entry across the basolateral membrane plays an important role in MD Ca2+ signaling.
It remains to be shown that MD Ca2+ signaling is directly involved in TGF signal transmission. However, because of the nature of the cell signaling properties of this divalent ion, we believe that it is very likely that MD [Ca2+]c plays a crucial role in TGF signaling. Ca2+ could subserve this function by activating transport processes, or by stimulating the release of a chemical mediator, or through some other signaling cascade that has yet to be discovered. Thus these studies suggest that the coupling between transport-related events at the apical membrane and the signal propagation by MD cells may involve an MD Ca2+ signaling system.
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ACKNOWLEDGEMENTS |
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We thank Martha Yeager for secretarial assistance.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-32032. J. Peti-Peterdi is a postdoctoral fellow from the Institute of Pathophysiology, Semmelweis University Medical School, Budapest, Hungary, and was supported by the Soros Foundation; he is currently supported by a postdoctural fellowship from the National Kidney Foundation.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. D. Bell, UAB Station, 865 Sparks Center, Univ. of Alabama at Birmingham, Birmingham, AL 35294 (E-mail: dbell{at}nrtc.dom.uab.edu).
Received 8 February 1999; accepted in final form 30 June 1999.
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