Articles in PresS. Am J Physiol Renal Physiol (January 28, 2015). doi:10.1152/ajprenal.00601.2014
1
Regulation of Glomerulotubular Balance III: Implication of Cytosolic Calcium in Flow-
2
dependent Proximal Tubule Transport
3 Zhaopeng Du1, Sheldon Weinbaum2, Alan M. Weinstein3 and Tong Wang1
4 5 6
1
Department of Cellular and Molecular Physiology, Yale University, New Haven, CT
7
2
Department of Biomedical Engineering, City College of New York, CUNY NY
8
3
Department of Physiology and Biophysics, Weill Medical College of Cornell University, NY
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Running title: Flow-dependent Proximal Tubule Transport
Address correspondence and reprint requests: Dr. Tong Wang Department of Cellular and Molecular Physiology Yale School of Medicine 333 Cedar Street, P.O. Box 208026 New Haven, CT 06520-8026 Phone: 203-785-6435 Fax: 203-785-4951 E-mail: [email protected]
Key words: Calcium Signals, Tubule transport, flow-dependent, Sodium Bicarbonate, IP3 receptor, extracellular Ca2+.
32 33
1 Copyright © 2015 by the American Physiological Society.
34
ABSTRACT
35 36
In proximal tubule, axial flow (drag on brush border microvilli) stimulates Na+ and HCO3-
37
reabsorption by modulating both NHE3 and H-ATPase activity, a process critical to
38
glomerulotubular balance. We have also demonstrated that blocking the angiotensin II receptor
39
decreases baseline transport, but preserves the flow effect; dopamine leaves baseline fluxes
40
intact, but abrogates the flow-effect. In the current work, we provide evidence implicating
41
cytosolic calcium in flow-dependent transport. Mouse proximal tubules were microperfused in
42
vitro at perfusion rates of 5 and 20 nl/min, and reabsorption of fluid (Jv) and HCO3- (JHCO3) were
43
measured. We examined the effect of high luminal Ca2+ (5 mM), 0 mM Ca2+, the Ca2+ chelator
44
BAPTA-AM, the IP3 receptor antagonist 2-APB, and the Ca-ATPase inhibitor thapsigargin. In
45
control tubules, increasing perfusion rate from 5 to 20 nl/min increased Jv by 62% and JHCO3 by
46
104%. With respect to Na+ reabsorption, high luminal Ca2+ decreased transport at low flow, but
47
preserved the flow-induced increase; low lumen Ca2+ had little impact; both BAPTA and 2-APB
48
had no effect on baseline flux, but abrogated the flow effect; thapsigargin decreased baseline
49
flow, leaving the flow effect intact. With respect to HCO3- reabsorption, high luminal Ca2+
50
decreased transport at low flow, and mildly diminished the flow-induced increase; low lumen
51
Ca2+ had little impact; both BAPTA and 2-APB had no effect on baseline flux, but abrogated the
52
flow effect. These data implicate IP3 receptor-mediated intracellular Ca2+ signaling as a critical
53
step in transduction of microvillous drag to modulate Na+ and HCO3- transport.
54
2
55 56
INTRODUCTION
57
Glomerulotubular balance (GTB) was first established in rat kidney via micropuncture
58
(18), where variation in glomerular filtration (over a range of flows from 15 to 60 nl/min) was
59
accompanied by a constant fractional proximal reabsorption. We have studied the mechanism of
60
axial flow induced changes in Na+ and HCO3- absorption by microperfusion of mouse proximal
61
tubules in vitro under low and high flow rates. From these studies we have demonstrated that the
62
underlying mechanism of GTB is the flow regulation of both NHE3 and H-ATPase activities.
63
This modulation is torque-dependent (bending moment at the apical membrane due to fluid flow)
64
and requires the intact actin cytoskeleton (2). In the studies of mouse proximal tubule cells, we
65
have shown that fluid shear stress stimulates NHE3 and H-ATPase trafficking to the apical and
66
Na/K-ATPase to the basolateral membrane surfaces (6); this observation is supported by the
67
mathematical model that shows both apical and basolateral transporters are regulated by flow
68
(25). Regulatory mechanisms of the GTB, including the role of dopamine and Ang II receptors
69
and the role of cAMP-PKA have been examined. From these studies we have demonstrated that
70
dopamine completely blocks flow-stimulated Na+ transport and partially abolishes flow-
71
stimulated HCO3- absorption, and the remaining portion of the increment of JHCO3 by flow is
72
blocked by the H-ATPase inhibitor bafilomycin. The effects of dopamine were completely
73
abolished by the DA1 receptor antagonist Sch 23390 (5). We also demonstrated a dopamine-
74
dependent decrease in the sensitivity of the renal brush border to a change in microvillous
75
torque; a DA1 antagonist increased the transport sensitivity to torque. These results are
76
consistent with the hypothesis that increased flow causes redistribution of NHE3 within the
77
apical membrane, as we have reported, in the proximal tubule cells (6); this activation is essential
78
for flow-stimulated Na+ and HCO3- absorption. The roles of Angiotensin II receptors (AT1a, 3
79
AT2) in flow effects in the proximal tubule were studied in WT and AT1a knockout mice. We
80
found that blocking the AT1 receptor by Losartan reduced absolute Na+ and HCO3- absorption at
81
both low and high flows, but did not affect fractional flow-stimulated transport. Compared with
82
WT control, in AT1a knockout (KO) mouse tubules, 53% of flow-stimulated Na+ absorption was
83
abolished, but flow-stimulated HCO3- absorption was retained at similar levels. The remaining
84
flow-stimulated HCO3- absorption was eliminated by the H-ATPase inhibitor bafilomycin.
85
Inhibition of the AT2 receptor by PD123319 increased both Na+ and HCO3- absorption, but did
86
not affect flow-mediated fractional changes. We also found that NHE3 expression at the protein
87
level was reduced in AT1a KO mice kidneys. These results indicate that the AT1 receptor is
88
important to maximize the NHE activity activated by flow; however, it is not critical for the flow
89
stimulated HCO3- transport, which still exists when the inhibitors are present or when the AT1a
90
receptor is knocked out (3).
91
Calcium signals are the major second messenger mechanisms for primary cilium-
92
mediated mechanosensation in MDCK cells (16) in cortical collecting duct (28) and in kidney
93
epithelial cells (15). However, regulation of flow effect by calcium signals in the proximal
94
tubule has never been studied. In the collecting duct, flow-stimulated K+ secretion is dependent
95
on apical calcium entry; removal of Ca2+ from the luminal perfusate prevents luminal Ca2+ entry
96
and abrogates flow stimulation of net K+ secretion (11). It is not known whether proximal tubule
97
cells response of Ca2+ is similar to other cell types. In the proximal tubule, however, it is
98
generally believed that an increase in cytosolic Ca2+ is associated with a decrease in NHE3-
99
mediated Na+ reabsorption (23) . It has been suggested that in the proximal tubule, cytosolic Ca2+
100
functions as a negative feedback signal at the luminal membrane to blunt Na+ entry (8, 27). In
101
the current study, we examine the effect of low and high luminal Ca2+, the Ca2+ chelator
102
BAPTA-AM, the IP3 receptor antagonist 2-APB, and the Ca-ATPase inhibitor thapsigargin on 4
103
flow stimulated Na+ and HCO3- absorption to investigate the role of calcium signals in flow-
104
activated proximal tubule transport. We demonstrate that the IP3 receptor-mediated Ca2+ signal is
105
crucial to flow-mediated Na+ and HCO3- absorption in proximal tubules.
106
5
107 108
METHODS Animals:
109
All experiments from animal work were conducted according to an Institutional Animal
110
Care and Use Committee-approved protocol at Yale School of Medicine. Mice, C57BL/6
111
purchased from the Jackson Lab, were maintained on a normal diet and tap water until the day of
112
the experiment and were anesthetized with intraperitoneal sodium pentobarbital (100 -150mg/kg)
113
at the time of the experiment. Both age (5 to 8 weeks) and gender (female) were matched among
114
control and all experimental groups.
115
Microperfusion of proximal tubules:
116
Kidneys were removed from the mouse after it was deeply anesthetized, and then
117
sectioned into ~1 mm thick slices. The slices were transferred to a 4oC HEPES-buffered Ringer’s
118
solution at pH of 7.4. Proximal convoluted tubules (S2 segments) were isolated by
119
microdissection in cooled (4oC) Hanks' solution, then transferred to the stage of an inverted
120
microscope, and cannulated using a series of concentric glass capillaries. The tubules were
121
perfused with an ultrafiltrate-like solution as described previously (4). The bath solution contains
122
similar electrolytes as the luminal solution, with added 3g/dl albumin. The perfusate and bath
123
solutions were bubbled with 95%O2-5%CO2, the pH was adjusted to 7.4, and the osmolalities to
124
300 mosmol/KgH2O in both solutions. All tubules were perfused at 37°C in a 1.2 ml
125
temperature-controlled bath at a flow rate of either 5nl/min or 20 nl/min, followed by three
126
sample collections for measuring 3H-inulin and HCO3- concentrations (4). The order of low or
127
high flow rate was used randomly. Bath fluid was continuously changed at a rate of 0.5 ml/min
128
to maintain the constancy of pH and bath osmolality. Extensively dialyzed [3H]-methoxy-inulin
129
was added to the perfusate at a concentration of 30 μCi/ml as a volume marker.
130
BAPTA-AM (10-7 M), the IP3 receptor antagonist 2-APB (10-7 M), and the Ca-ATPase 6
131
inhibitor thapsigargin (10-7 M) were added to the luminal perfusate, and were all purchased from
132
Sigma (St. Louis, MO).
133
Sample measurement and analysis:
134
The rates of fluid (Jv) and HCO3- (JHCO3 ) absorption were calculated by measuring the
135
concentrations of 3H-inulin and total CO2, as described previously (4). A constant-bore glass
136
capillary was used to obtain precise aliquots of initial perfusates and collection of samples to be
137
analyzed for [3H]-methoxy-inulin by liquid scintillation spectroscopy. The total CO2
138
concentration of both initial and collected fluids was measured by the nanoflow spectrometer
139
(WPI). The rates of net fluid and HCO3- absorption was calculated as described previously and
140
expressed per mm tubular length (4).
141
The JNa was calculated according to the rate of fluid absorption ([Na] * Jv), since the ratio
142
of fluid and Na+ absorption is 1 in the proximal tubule (24). The JCl was calculated as JCl = JNa –
143
JHCO3. The cell volume is identified with epithelial volume, and is calculated as: [π•(OD/2)2 -
144
π•(ID/2)2 ], where ID is inner tubular diameter, and OD is outer tubule diameter. In this
145
estimate, it is assumed that lateral intercellular space volume is a negligible fraction of epithelial
146
volume. In this regard, Tisher and Kokko (20) determined that in rabbit proximal tubule, the
147
spacing between membranes of opposing cells is about 0.03 micron at baseline, and grows or
148
shrinks 10% with changes in transport.
149
proximal tubule, whose area is expanded 20-fold over a simple cylinder (26), interspace volume
150
is estimated to be less than 5% of epithelial volume.
151 152
Thus, even with the pleated lateral membrane of
The total torque (bending moment) T on the microvilli due to fluid flow was calculated by the equation which we have published previously (2).
7
L + δ L2 1+ + 2 T Rr2 R 2R μ Q = 2 Tr R L + δ L2 μr Qr + 2 1+ Rr 2Rr
r—index to reference value R—inner tubule radius with brush border L— length of the microvilli (L = 2.5 μm) δ − microvilli tip interaction layer (δ=150 nm) μ —fluid viscosity Q— flow rate in the tubule
153 154 155 156
Applying this formula to the experiments under consideration, variations in microvillous length,
157
L, of 50% or less should produce inaccuracies in the torque ratio of less than 10%. That height is
158
not measured in these experiments, and is assumed constant in the torque estimates.
159
Statistics:
160
Data are presented as means ± SE. Student’s t-test was used to compare control and experimental
161
groups. ANOVA test was used for comparison of several experimental groups with a control
162
group following by Dunnett’s test. The difference between the mean values of an experimental
163
group and a control group was considered significant if P < 0.05.
164 165
8
166 167
RESULTS Impact of extracellular and intracellular Ca2+ depletion on flow-mediated tubule transport:
168
The effects of extracellular Ca2+ depletion on flow-induced changes in Na+ and HCO3-
169
absorption were examined by microperfusion of proximal tubules with low and high flow rates.
170
The extracellular Ca2+ depletion was achieved by using Ca2+ free solution plus 1mM of EGTA
171
(cell-impermeant calcium chelator) in the luminal perfusate. Under this condition, the
172
extracellular Ca2+ in the apical side is nearly 0 mM. We have compared the flow-induced
173
changes in fluid (Jv), Na+ (JNa) and HCO3- (JHCO3) absorption between the luminal perfusate with
174
1mM Ca2+ (control group) and with 0mM Ca2+ plus 1mM EGTA. As we previously observed, an
175
increase in perfusion rate from 5 to 20 nl/min increased Jv , JNa by 62% and JHCO3 by 104% in
176
control group (Fig. 1). Depletion of extracellular Ca2+ had no effect on Jv , JNa or JHCO3 under
177
either low or high flow conditions. The fractional changes in fluid, Na+, HCO3- and calculated
178
Cl- absorption (JCl) by flow are also the same in control and extracellular Ca2+ depletion groups
179
(Figs. 1 and 3). This finding indicates that luminal Ca2+ influx does not mediate flow-stimulated
180
proximal tubule transport.
181
We next examined the effects of both intracellular and extracellular Ca2+ depletion on
182
flow-induced changes in Na+ and HCO3- absorption. Complete Ca2+ depletion was achieved by
183
using Ca2+ free solution plus EGTA and a cell-permeant Ca2+ chelator BAPTA-AM (10-7M) in
184
the luminal perfusate (11). Under this condition, both extracellular and intracellular Ca2+ are
185
depleted. We have compared the flow-induced changes in Jv, JNa JCl and JHCO3 between the
186
luminal perfusate with 1mM Ca2+ and with 0 mM Ca2+ plus EGTA and BAPTA-AM. As shown
187
in Fig. 2, an increase in perfusion rate from 5 to 20 nl/min JNa and Jv increased by 62% and JHCO3
188
increased by 104% in control group. Depletion of both extracellular and intracellular Ca2+ did
189
not change Na+ or HCO3- absorption at the low flow rate. In contrast, flow stimulated Na+ 9
190
absorption was completely abrogated and flow stimulated HCO3- absorption was significantly
191
reduced in the Ca2+depletion group compared with the control. The fractional change of JNa and
192
Jv by flow was 62.3% and -22.7% (P
1
Regulation of Glomerulotubular Balance III: Implication of Cytosolic Calcium in Flow-
2
dependent Proximal Tubule Transport
3 Zhaopeng Du1, Sheldon Weinbaum2, Alan M. Weinstein3 and Tong Wang1
4 5 6
1
Department of Cellular and Molecular Physiology, Yale University, New Haven, CT
7
2
Department of Biomedical Engineering, City College of New York, CUNY NY
8
3
Department of Physiology and Biophysics, Weill Medical College of Cornell University, NY
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Running title: Flow-dependent Proximal Tubule Transport
Address correspondence and reprint requests: Dr. Tong Wang Department of Cellular and Molecular Physiology Yale School of Medicine 333 Cedar Street, P.O. Box 208026 New Haven, CT 06520-8026 Phone: 203-785-6435 Fax: 203-785-4951 E-mail: [email protected]
Key words: Calcium Signals, Tubule transport, flow-dependent, Sodium Bicarbonate, IP3 receptor, extracellular Ca2+.
32 33
1 Copyright © 2015 by the American Physiological Society.
34
ABSTRACT
35 36
In proximal tubule, axial flow (drag on brush border microvilli) stimulates Na+ and HCO3-
37
reabsorption by modulating both NHE3 and H-ATPase activity, a process critical to
38
glomerulotubular balance. We have also demonstrated that blocking the angiotensin II receptor
39
decreases baseline transport, but preserves the flow effect; dopamine leaves baseline fluxes
40
intact, but abrogates the flow-effect. In the current work, we provide evidence implicating
41
cytosolic calcium in flow-dependent transport. Mouse proximal tubules were microperfused in
42
vitro at perfusion rates of 5 and 20 nl/min, and reabsorption of fluid (Jv) and HCO3- (JHCO3) were
43
measured. We examined the effect of high luminal Ca2+ (5 mM), 0 mM Ca2+, the Ca2+ chelator
44
BAPTA-AM, the IP3 receptor antagonist 2-APB, and the Ca-ATPase inhibitor thapsigargin. In
45
control tubules, increasing perfusion rate from 5 to 20 nl/min increased Jv by 62% and JHCO3 by
46
104%. With respect to Na+ reabsorption, high luminal Ca2+ decreased transport at low flow, but
47
preserved the flow-induced increase; low lumen Ca2+ had little impact; both BAPTA and 2-APB
48
had no effect on baseline flux, but abrogated the flow effect; thapsigargin decreased baseline
49
flow, leaving the flow effect intact. With respect to HCO3- reabsorption, high luminal Ca2+
50
decreased transport at low flow, and mildly diminished the flow-induced increase; low lumen
51
Ca2+ had little impact; both BAPTA and 2-APB had no effect on baseline flux, but abrogated the
52
flow effect. These data implicate IP3 receptor-mediated intracellular Ca2+ signaling as a critical
53
step in transduction of microvillous drag to modulate Na+ and HCO3- transport.
54
2
55 56
INTRODUCTION
57
Glomerulotubular balance (GTB) was first established in rat kidney via micropuncture
58
(18), where variation in glomerular filtration (over a range of flows from 15 to 60 nl/min) was
59
accompanied by a constant fractional proximal reabsorption. We have studied the mechanism of
60
axial flow induced changes in Na+ and HCO3- absorption by microperfusion of mouse proximal
61
tubules in vitro under low and high flow rates. From these studies we have demonstrated that the
62
underlying mechanism of GTB is the flow regulation of both NHE3 and H-ATPase activities.
63
This modulation is torque-dependent (bending moment at the apical membrane due to fluid flow)
64
and requires the intact actin cytoskeleton (2). In the studies of mouse proximal tubule cells, we
65
have shown that fluid shear stress stimulates NHE3 and H-ATPase trafficking to the apical and
66
Na/K-ATPase to the basolateral membrane surfaces (6); this observation is supported by the
67
mathematical model that shows both apical and basolateral transporters are regulated by flow
68
(25). Regulatory mechanisms of the GTB, including the role of dopamine and Ang II receptors
69
and the role of cAMP-PKA have been examined. From these studies we have demonstrated that
70
dopamine completely blocks flow-stimulated Na+ transport and partially abolishes flow-
71
stimulated HCO3- absorption, and the remaining portion of the increment of JHCO3 by flow is
72
blocked by the H-ATPase inhibitor bafilomycin. The effects of dopamine were completely
73
abolished by the DA1 receptor antagonist Sch 23390 (5). We also demonstrated a dopamine-
74
dependent decrease in the sensitivity of the renal brush border to a change in microvillous
75
torque; a DA1 antagonist increased the transport sensitivity to torque. These results are
76
consistent with the hypothesis that increased flow causes redistribution of NHE3 within the
77
apical membrane, as we have reported, in the proximal tubule cells (6); this activation is essential
78
for flow-stimulated Na+ and HCO3- absorption. The roles of Angiotensin II receptors (AT1a, 3
79
AT2) in flow effects in the proximal tubule were studied in WT and AT1a knockout mice. We
80
found that blocking the AT1 receptor by Losartan reduced absolute Na+ and HCO3- absorption at
81
both low and high flows, but did not affect fractional flow-stimulated transport. Compared with
82
WT control, in AT1a knockout (KO) mouse tubules, 53% of flow-stimulated Na+ absorption was
83
abolished, but flow-stimulated HCO3- absorption was retained at similar levels. The remaining
84
flow-stimulated HCO3- absorption was eliminated by the H-ATPase inhibitor bafilomycin.
85
Inhibition of the AT2 receptor by PD123319 increased both Na+ and HCO3- absorption, but did
86
not affect flow-mediated fractional changes. We also found that NHE3 expression at the protein
87
level was reduced in AT1a KO mice kidneys. These results indicate that the AT1 receptor is
88
important to maximize the NHE activity activated by flow; however, it is not critical for the flow
89
stimulated HCO3- transport, which still exists when the inhibitors are present or when the AT1a
90
receptor is knocked out (3).
91
Calcium signals are the major second messenger mechanisms for primary cilium-
92
mediated mechanosensation in MDCK cells (16) in cortical collecting duct (28) and in kidney
93
epithelial cells (15). However, regulation of flow effect by calcium signals in the proximal
94
tubule has never been studied. In the collecting duct, flow-stimulated K+ secretion is dependent
95
on apical calcium entry; removal of Ca2+ from the luminal perfusate prevents luminal Ca2+ entry
96
and abrogates flow stimulation of net K+ secretion (11). It is not known whether proximal tubule
97
cells response of Ca2+ is similar to other cell types. In the proximal tubule, however, it is
98
generally believed that an increase in cytosolic Ca2+ is associated with a decrease in NHE3-
99
mediated Na+ reabsorption (23) . It has been suggested that in the proximal tubule, cytosolic Ca2+
100
functions as a negative feedback signal at the luminal membrane to blunt Na+ entry (8, 27). In
101
the current study, we examine the effect of low and high luminal Ca2+, the Ca2+ chelator
102
BAPTA-AM, the IP3 receptor antagonist 2-APB, and the Ca-ATPase inhibitor thapsigargin on 4
103
flow stimulated Na+ and HCO3- absorption to investigate the role of calcium signals in flow-
104
activated proximal tubule transport. We demonstrate that the IP3 receptor-mediated Ca2+ signal is
105
crucial to flow-mediated Na+ and HCO3- absorption in proximal tubules.
106
5
107 108
METHODS Animals:
109
All experiments from animal work were conducted according to an Institutional Animal
110
Care and Use Committee-approved protocol at Yale School of Medicine. Mice, C57BL/6
111
purchased from the Jackson Lab, were maintained on a normal diet and tap water until the day of
112
the experiment and were anesthetized with intraperitoneal sodium pentobarbital (100 -150mg/kg)
113
at the time of the experiment. Both age (5 to 8 weeks) and gender (female) were matched among
114
control and all experimental groups.
115
Microperfusion of proximal tubules:
116
Kidneys were removed from the mouse after it was deeply anesthetized, and then
117
sectioned into ~1 mm thick slices. The slices were transferred to a 4oC HEPES-buffered Ringer’s
118
solution at pH of 7.4. Proximal convoluted tubules (S2 segments) were isolated by
119
microdissection in cooled (4oC) Hanks' solution, then transferred to the stage of an inverted
120
microscope, and cannulated using a series of concentric glass capillaries. The tubules were
121
perfused with an ultrafiltrate-like solution as described previously (4). The bath solution contains
122
similar electrolytes as the luminal solution, with added 3g/dl albumin. The perfusate and bath
123
solutions were bubbled with 95%O2-5%CO2, the pH was adjusted to 7.4, and the osmolalities to
124
300 mosmol/KgH2O in both solutions. All tubules were perfused at 37°C in a 1.2 ml
125
temperature-controlled bath at a flow rate of either 5nl/min or 20 nl/min, followed by three
126
sample collections for measuring 3H-inulin and HCO3- concentrations (4). The order of low or
127
high flow rate was used randomly. Bath fluid was continuously changed at a rate of 0.5 ml/min
128
to maintain the constancy of pH and bath osmolality. Extensively dialyzed [3H]-methoxy-inulin
129
was added to the perfusate at a concentration of 30 μCi/ml as a volume marker.
130
BAPTA-AM (10-7 M), the IP3 receptor antagonist 2-APB (10-7 M), and the Ca-ATPase 6
131
inhibitor thapsigargin (10-7 M) were added to the luminal perfusate, and were all purchased from
132
Sigma (St. Louis, MO).
133
Sample measurement and analysis:
134
The rates of fluid (Jv) and HCO3- (JHCO3 ) absorption were calculated by measuring the
135
concentrations of 3H-inulin and total CO2, as described previously (4). A constant-bore glass
136
capillary was used to obtain precise aliquots of initial perfusates and collection of samples to be
137
analyzed for [3H]-methoxy-inulin by liquid scintillation spectroscopy. The total CO2
138
concentration of both initial and collected fluids was measured by the nanoflow spectrometer
139
(WPI). The rates of net fluid and HCO3- absorption was calculated as described previously and
140
expressed per mm tubular length (4).
141
The JNa was calculated according to the rate of fluid absorption ([Na] * Jv), since the ratio
142
of fluid and Na+ absorption is 1 in the proximal tubule (24). The JCl was calculated as JCl = JNa –
143
JHCO3. The cell volume is identified with epithelial volume, and is calculated as: [π•(OD/2)2 -
144
π•(ID/2)2 ], where ID is inner tubular diameter, and OD is outer tubule diameter. In this
145
estimate, it is assumed that lateral intercellular space volume is a negligible fraction of epithelial
146
volume. In this regard, Tisher and Kokko (20) determined that in rabbit proximal tubule, the
147
spacing between membranes of opposing cells is about 0.03 micron at baseline, and grows or
148
shrinks 10% with changes in transport.
149
proximal tubule, whose area is expanded 20-fold over a simple cylinder (26), interspace volume
150
is estimated to be less than 5% of epithelial volume.
151 152
Thus, even with the pleated lateral membrane of
The total torque (bending moment) T on the microvilli due to fluid flow was calculated by the equation which we have published previously (2).
7
L + δ L2 1+ + 2 T Rr2 R 2R μ Q = 2 Tr R L + δ L2 μr Qr + 2 1+ Rr 2Rr
r—index to reference value R—inner tubule radius with brush border L— length of the microvilli (L = 2.5 μm) δ − microvilli tip interaction layer (δ=150 nm) μ —fluid viscosity Q— flow rate in the tubule
153 154 155 156
Applying this formula to the experiments under consideration, variations in microvillous length,
157
L, of 50% or less should produce inaccuracies in the torque ratio of less than 10%. That height is
158
not measured in these experiments, and is assumed constant in the torque estimates.
159
Statistics:
160
Data are presented as means ± SE. Student’s t-test was used to compare control and experimental
161
groups. ANOVA test was used for comparison of several experimental groups with a control
162
group following by Dunnett’s test. The difference between the mean values of an experimental
163
group and a control group was considered significant if P < 0.05.
164 165
8
166 167
RESULTS Impact of extracellular and intracellular Ca2+ depletion on flow-mediated tubule transport:
168
The effects of extracellular Ca2+ depletion on flow-induced changes in Na+ and HCO3-
169
absorption were examined by microperfusion of proximal tubules with low and high flow rates.
170
The extracellular Ca2+ depletion was achieved by using Ca2+ free solution plus 1mM of EGTA
171
(cell-impermeant calcium chelator) in the luminal perfusate. Under this condition, the
172
extracellular Ca2+ in the apical side is nearly 0 mM. We have compared the flow-induced
173
changes in fluid (Jv), Na+ (JNa) and HCO3- (JHCO3) absorption between the luminal perfusate with
174
1mM Ca2+ (control group) and with 0mM Ca2+ plus 1mM EGTA. As we previously observed, an
175
increase in perfusion rate from 5 to 20 nl/min increased Jv , JNa by 62% and JHCO3 by 104% in
176
control group (Fig. 1). Depletion of extracellular Ca2+ had no effect on Jv , JNa or JHCO3 under
177
either low or high flow conditions. The fractional changes in fluid, Na+, HCO3- and calculated
178
Cl- absorption (JCl) by flow are also the same in control and extracellular Ca2+ depletion groups
179
(Figs. 1 and 3). This finding indicates that luminal Ca2+ influx does not mediate flow-stimulated
180
proximal tubule transport.
181
We next examined the effects of both intracellular and extracellular Ca2+ depletion on
182
flow-induced changes in Na+ and HCO3- absorption. Complete Ca2+ depletion was achieved by
183
using Ca2+ free solution plus EGTA and a cell-permeant Ca2+ chelator BAPTA-AM (10-7M) in
184
the luminal perfusate (11). Under this condition, both extracellular and intracellular Ca2+ are
185
depleted. We have compared the flow-induced changes in Jv, JNa JCl and JHCO3 between the
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luminal perfusate with 1mM Ca2+ and with 0 mM Ca2+ plus EGTA and BAPTA-AM. As shown
187
in Fig. 2, an increase in perfusion rate from 5 to 20 nl/min JNa and Jv increased by 62% and JHCO3
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increased by 104% in control group. Depletion of both extracellular and intracellular Ca2+ did
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not change Na+ or HCO3- absorption at the low flow rate. In contrast, flow stimulated Na+ 9
190
absorption was completely abrogated and flow stimulated HCO3- absorption was significantly
191
reduced in the Ca2+depletion group compared with the control. The fractional change of JNa and
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Jv by flow was 62.3% and -22.7% (P
