Acta Physiol 2015, 214, 319–328
TRPV4 mediates flow-induced increases in intracellular Ca in medullary thick ascending limbs P. D. Cabral,1,2 C. Capurro2 and J. L. Garvin1 1 Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, USA 2 Facultad de Medicina, IFIBIO–HOUSSAY, UBA-CONICET, Departamento de Ciencias Fisiologicas, Ciudad Autonoma de Buenos Aires, Universidad de Buenos Aires, Buenos Aires, Argentina
Received 3 December 2014, revision requested 8 January 2015, revision received 15 April 2015, accepted 12 May 2015 Correspondence: P. D. Cabral, Department of Physiology and Biophysics, Case Western Reserve University, 10900 Euclid Avenue, Robbins-E532, Cleveland, OH 44106, USA. E-mail: [email protected]
Abstract Medullary thick ascending limbs (mTAL) regulate Na balance and therefore blood pressure. We previously showed that cell swelling and luminal flow activates the mechanosensitive channel TRPV4 in mTAL. Aim: We hypothesized that TRPV4 mediates flow-induced increases in intracellular Ca (Cai) in rat mTALs. Methods: We performed ratiometric measurements of Cai in perfused mTALs. Results: Increasing luminal flow from 0 to 20 nL min1 caused Cai to peak 231 29 nmol L1 above basal concentrations (n = 18). The general TRPV inhibitor ruthenium red at 15 and 50 lmol L1 reduced peak Cai by 41 9 (P < 0.01; n = 5) and 77 10% (P < 0.02; n = 6). The selective TRPV4 inhibitor RN1734 at 10 and 50 lmol L1 reduced peak Cai by 46 11 (P < 0.01; n = 7) and 76 5% (P < 0.02; n = 5) respectively. To specifically target TRPV4, mTALs were transduced with adenoviruses expressing TRPV4 small hairpin (sh) RNA. In non-transduced control mTALs, luminal flow generated a peak increase in Cai of 111 21 nmol L1 (n = 8). In TRPV4shRNA-transduced mTALs, the Cai peak was reduced to 56 8 nmol L1 (P < 0.03, n = 9). Removing extracellular Ca completely abolished flow-induced increases in Cai. Increasing luminal flow in the presence of hexokinase 20 (U mL1) to scavenge extracellular ATP did not modify significantly the increases in Cai induced by luminal flow. Finally, we studied the effect of the TRPV4 selective agonist GSK1016790A on Cai. In the absence of luminal flow, GSK1016790A (10 nmol L1) increased Cai from 60 11 nmol L1 to 262 71 nmol L1 (P < 0.05; n = 7). Conclusion: We conclude that flow-induced increases in Cai are mediated primarily by TRPV4 in the rat mTAL. Keywords mechanosensation, nitric oxide, TRP channels.
The thick ascending limb of the loop of Henle (TAL) plays a critical role in salt homoeostasis and blood pressure regulation because it reabsorbs approx. 30% of the NaCl filtered by the glomerulus (Burg 1982). This is best evidenced by the fact that pathologies that cause elevated TAL NaCl reabsorption increase blood
pressure while those that reduce it cause hypotension (Hebert 2003, Jung et al. 2011). Luminal flow through the nephron is not static; rather it constantly changes. Acutely, flow changes due to tubuloglomerular feedback and peristalsis of the renal pelvis (Holstein-Rathlou & Marsh 1990,
© 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12528
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TRPV4 mediates flow-induced TAL Cai2+ increase
· P D Cabral et al.
Dwyer & Schmidt-Nielsen 2003). Increases in volume and salt intake also rapidly enhance luminal flow (Arendshorst & Beierwaltes 1979, Mozaffari et al. 1991). Hypertension and glomerular hyperfiltration in the early stages of diabetes chronically elevate luminal flow (DiBona & Rios 1978, Pollock et al. 1991). Increases in luminal flow have been shown to augment nitric oxide and superoxide production, and ATP release from TALs (Cabral et al. 2010, 2012, Cabral & Garvin 2011). They have also been shown to enhance K secretion and Na reabsorption in the collecting duct (Woda et al. 2001, 2003, Morimoto et al. 2006). Increasing luminal flow has been shown to elevate intracellular Ca (Cai) in both thick ascending limbs and collecting ducts (Woda et al. 2002, Liu et al. 2003, Jensen et al. 2007). This is thought to be the initiating signal for flow-induced changes in different physiological parameters such as ionic transport in both nephron segments. However, the transporters mediating the flow-induced increase in Cai are not well understood. The transient receptor potential vanilloid 4 (TRPV4) channel, a member of the TRPV family of cation channels, displays mechanosensitive activation (O’Neil & Heller 2005, Liedtke 2007). It transduces cellular stretch and flow-induced shear stress into Ca influx increasing Cai in vascular smooth muscle and endothelial cells (Kohler et al. 2006, Mendoza et al. 2010, Filosa et al. 2013). In the nephron, Berrout et al. (2012) showed that TRPV4 mediates flow-induced Cai increases in the mouse collecting duct. We have previously shown that TRPV4 is expressed in the rat TAL and mediates cell swellinginduced ATP release in this segment (Silva & Garvin 2008, Cabral & Garvin 2014); however, it is not known whether this channel mediates the increase in Cai caused by luminal flow in this segment. We hypothesized that TRPV4 mediates flow-induced elevation of Cai in rat TALs.
Materials and methods
Acta Physiol 2015, 214, 319–328
Solutions and chemicals Fura 2-AM and 4-Bromo A-23187 were purchased from Invitrogen (Eugene, OR, USA). Ruthenium red, hexokinase, GSK1016790A and EGTA (ethylene glycol-bis(b-aminoethyl ether)-N,N,N0 ,N0 -tetraacetic acid tetrasodium salt) were obtained from Sigma-Aldrich (St. Louis, MO) and RN1734 from Tocris Bioscience (Bristol, UK). The composition of the physiological saline used to perfuse and bathe the tubules was (in mmol L1) as follows: 130 NaCl, four KCl, mix of monobasic and dibasic phosphate, 1.2 MgSO4, 6 Lalanine, 0.1 L-arginine, one trisodium citrate, 5.5 glucose, two calcium dilactate, and 10 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), pH 7.4 at 37 °C. The calcium-free solution was (in mmol L1): 130 NaCl, four KCl, 1.2 MgSO4, 6 L-alanine, 0.1 Larginine, 5.5 glucose, 0.1 ethylene glycol tetraacetic acid (EGTA) and 10 4-(2-hydroxyethyl) piperazine-1ethanesulfonic acid (HEPES), pH 7.4 at 37 °C. Solutions were adjusted to 290 3 mOsm kg1 H2O with NaCl as necessary as measured by freezing-point depression.
Preparation of isolated and perfused medullary thick ascending limbs Medullary TALs were isolated and perfused as previously described. Briefly, rats were anesthetized with ketamine (100 mg kg1 body wt i.p.) and xylazine (20 mg kg1 body wt i.p.). The abdominal cavity was opened and the left kidney was superfused with icecold 150 mmol L1 NaCl, then removed and placed in physiological saline (4 °C). Coronal slices were cut and TALs isolated from the outer medulla under a stereomicroscope at 4–10 °C. TALs ranging from 0.7 to 1.0 mm were transferred to a temperature-regulated chamber and perfused using concentric glass pipettes at 37 1 °C. Luminal perfusion rates were 0 or 20 nL min1, and basolateral flow rate was 1 mL min1.
Animals
Measurements of Cai
Male Sprague-Dawley rats weighing 100–150 g from Charles River Breeding Laboratories, Wilmington, MA, were fed a diet containing 0.22% sodium and 1.1% potassium (Purina, Richmond, IN, USA) for at least 5 days prior to the experiments. All protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Case Western Reserve University following the National Institutes of Health Guidelines for Use and Care of Experimental Animals.
Isolated TALs were loaded with 1 lmol L1 FURA 2AM for 30 min and washed for 30 min using dye-free physiological saline at 37 1 °C. The TRPV4 antagonists (ruthenium red and RN1734) and the ATP scavenger hexokinase were present in both luminal and basolateral sides throughout the 30 min-washing period. FURA 2 was excited at 340 nm and 380 nm. The ratio between the emitted fluorescence of both wavelengths is proportional to Cai. Fluorescence was imaged digitally with an image intensifier and a
320
© 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12528
P D Cabral et al.
charge-coupled device camera. Fluorescence images were taken every 2-s in the absence and presence of luminal flow. Data were recorded using METAFLUOR version 7 imaging software (Universal Imaging, Downington, PA, USA). Cai was calibrated at the end of each experiment with 5 mmol L1 EGTA and 10 lmol L1 4-Br-A23187. Cai was calculated using the following equation as previously described: ½Cai ¼ Kd ðFmin =Fmax Þ ðR Rmin Þ=ðRmax RÞ where Kd is the Ca dissociation constant of FURA 2 (224 nmol L1); R is the ratio between 340 nm and 380 nm of each sample; Fmin is the fluorescence intensity of 380 nm during EGTA; Fmax is the fluorescence intensity of 380 nm during 4-Br-A23187; Rmin is the minimum ratio between 340 nm and 380 nm during EGTA, and Rmax is the maximum ratio between 340 nm and 380 nm during 4-Br-A23187. For each experiment where luminal flow was tested, we calculated and analysed the following variables: peak increase of Cai (nmol L1); rate of rise (nmol L1 s1); half-life of the decay (s); and the plateau of the decay (nmol L1). In the set of experiments where the TRPV4 agonist GSK1016790A was tested in the absence of luminal flow, the peak increase of Cai and the rate of rise were analysed.
In vivo adenoviral transduction of medullary thick ascending limbs Small hairpin TRPV4-RNA (shRNA)-expressing adenoviruses under the control of the H1 mouse RNA polymerase promoter were delivered to mTAL as previously described (Ortiz et al. 2003, Silva & Garvin 2008). Briefly, rats were anesthetized with ketamine (60 mg kg1 body wt i.p.) and xylazine (20 mg kg1 body wt i.p.). After anaesthesia, the left kidney was exposed through a flanked incision and the renal artery and vein clamped. Then, four infusions of 20 lL of adenovirus at a final concentration of 1.2 9 1012 particles per mL were injected at the corticomedullary boundary at a flow rate of 20 lL min1 using a 30-gauge needle and syringe pump. The needle was removed from the injection site 30 s after each infusion was completed to avoid bleeding and leakage of the virus. After a total time of 8 min, the vascular clamps were removed, the kidney returned to the abdomen and the wound sutured. After surgery, animals were kept warm and under direct observation until they recovered from the anaesthesia and appeared alert. Then, they were returned to their cages, where food and water were available ad libitum. Experiments were performed 72 h after surgery.
· TRPV4 mediates flow-induced TAL Cai
2+
increase
Statistical analysis Results are presented as mean standard error. An unpaired Student’s t-test was used to calculate the difference between no flow and flow conditions in separated experimental groups. A P value of
TRPV4 mediates flow-induced increases in intracellular Ca in medullary thick ascending limbs P. D. Cabral,1,2 C. Capurro2 and J. L. Garvin1 1 Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, USA 2 Facultad de Medicina, IFIBIO–HOUSSAY, UBA-CONICET, Departamento de Ciencias Fisiologicas, Ciudad Autonoma de Buenos Aires, Universidad de Buenos Aires, Buenos Aires, Argentina
Received 3 December 2014, revision requested 8 January 2015, revision received 15 April 2015, accepted 12 May 2015 Correspondence: P. D. Cabral, Department of Physiology and Biophysics, Case Western Reserve University, 10900 Euclid Avenue, Robbins-E532, Cleveland, OH 44106, USA. E-mail: [email protected]
Abstract Medullary thick ascending limbs (mTAL) regulate Na balance and therefore blood pressure. We previously showed that cell swelling and luminal flow activates the mechanosensitive channel TRPV4 in mTAL. Aim: We hypothesized that TRPV4 mediates flow-induced increases in intracellular Ca (Cai) in rat mTALs. Methods: We performed ratiometric measurements of Cai in perfused mTALs. Results: Increasing luminal flow from 0 to 20 nL min1 caused Cai to peak 231 29 nmol L1 above basal concentrations (n = 18). The general TRPV inhibitor ruthenium red at 15 and 50 lmol L1 reduced peak Cai by 41 9 (P < 0.01; n = 5) and 77 10% (P < 0.02; n = 6). The selective TRPV4 inhibitor RN1734 at 10 and 50 lmol L1 reduced peak Cai by 46 11 (P < 0.01; n = 7) and 76 5% (P < 0.02; n = 5) respectively. To specifically target TRPV4, mTALs were transduced with adenoviruses expressing TRPV4 small hairpin (sh) RNA. In non-transduced control mTALs, luminal flow generated a peak increase in Cai of 111 21 nmol L1 (n = 8). In TRPV4shRNA-transduced mTALs, the Cai peak was reduced to 56 8 nmol L1 (P < 0.03, n = 9). Removing extracellular Ca completely abolished flow-induced increases in Cai. Increasing luminal flow in the presence of hexokinase 20 (U mL1) to scavenge extracellular ATP did not modify significantly the increases in Cai induced by luminal flow. Finally, we studied the effect of the TRPV4 selective agonist GSK1016790A on Cai. In the absence of luminal flow, GSK1016790A (10 nmol L1) increased Cai from 60 11 nmol L1 to 262 71 nmol L1 (P < 0.05; n = 7). Conclusion: We conclude that flow-induced increases in Cai are mediated primarily by TRPV4 in the rat mTAL. Keywords mechanosensation, nitric oxide, TRP channels.
The thick ascending limb of the loop of Henle (TAL) plays a critical role in salt homoeostasis and blood pressure regulation because it reabsorbs approx. 30% of the NaCl filtered by the glomerulus (Burg 1982). This is best evidenced by the fact that pathologies that cause elevated TAL NaCl reabsorption increase blood
pressure while those that reduce it cause hypotension (Hebert 2003, Jung et al. 2011). Luminal flow through the nephron is not static; rather it constantly changes. Acutely, flow changes due to tubuloglomerular feedback and peristalsis of the renal pelvis (Holstein-Rathlou & Marsh 1990,
© 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12528
319
TRPV4 mediates flow-induced TAL Cai2+ increase
· P D Cabral et al.
Dwyer & Schmidt-Nielsen 2003). Increases in volume and salt intake also rapidly enhance luminal flow (Arendshorst & Beierwaltes 1979, Mozaffari et al. 1991). Hypertension and glomerular hyperfiltration in the early stages of diabetes chronically elevate luminal flow (DiBona & Rios 1978, Pollock et al. 1991). Increases in luminal flow have been shown to augment nitric oxide and superoxide production, and ATP release from TALs (Cabral et al. 2010, 2012, Cabral & Garvin 2011). They have also been shown to enhance K secretion and Na reabsorption in the collecting duct (Woda et al. 2001, 2003, Morimoto et al. 2006). Increasing luminal flow has been shown to elevate intracellular Ca (Cai) in both thick ascending limbs and collecting ducts (Woda et al. 2002, Liu et al. 2003, Jensen et al. 2007). This is thought to be the initiating signal for flow-induced changes in different physiological parameters such as ionic transport in both nephron segments. However, the transporters mediating the flow-induced increase in Cai are not well understood. The transient receptor potential vanilloid 4 (TRPV4) channel, a member of the TRPV family of cation channels, displays mechanosensitive activation (O’Neil & Heller 2005, Liedtke 2007). It transduces cellular stretch and flow-induced shear stress into Ca influx increasing Cai in vascular smooth muscle and endothelial cells (Kohler et al. 2006, Mendoza et al. 2010, Filosa et al. 2013). In the nephron, Berrout et al. (2012) showed that TRPV4 mediates flow-induced Cai increases in the mouse collecting duct. We have previously shown that TRPV4 is expressed in the rat TAL and mediates cell swellinginduced ATP release in this segment (Silva & Garvin 2008, Cabral & Garvin 2014); however, it is not known whether this channel mediates the increase in Cai caused by luminal flow in this segment. We hypothesized that TRPV4 mediates flow-induced elevation of Cai in rat TALs.
Materials and methods
Acta Physiol 2015, 214, 319–328
Solutions and chemicals Fura 2-AM and 4-Bromo A-23187 were purchased from Invitrogen (Eugene, OR, USA). Ruthenium red, hexokinase, GSK1016790A and EGTA (ethylene glycol-bis(b-aminoethyl ether)-N,N,N0 ,N0 -tetraacetic acid tetrasodium salt) were obtained from Sigma-Aldrich (St. Louis, MO) and RN1734 from Tocris Bioscience (Bristol, UK). The composition of the physiological saline used to perfuse and bathe the tubules was (in mmol L1) as follows: 130 NaCl, four KCl, mix of monobasic and dibasic phosphate, 1.2 MgSO4, 6 Lalanine, 0.1 L-arginine, one trisodium citrate, 5.5 glucose, two calcium dilactate, and 10 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), pH 7.4 at 37 °C. The calcium-free solution was (in mmol L1): 130 NaCl, four KCl, 1.2 MgSO4, 6 L-alanine, 0.1 Larginine, 5.5 glucose, 0.1 ethylene glycol tetraacetic acid (EGTA) and 10 4-(2-hydroxyethyl) piperazine-1ethanesulfonic acid (HEPES), pH 7.4 at 37 °C. Solutions were adjusted to 290 3 mOsm kg1 H2O with NaCl as necessary as measured by freezing-point depression.
Preparation of isolated and perfused medullary thick ascending limbs Medullary TALs were isolated and perfused as previously described. Briefly, rats were anesthetized with ketamine (100 mg kg1 body wt i.p.) and xylazine (20 mg kg1 body wt i.p.). The abdominal cavity was opened and the left kidney was superfused with icecold 150 mmol L1 NaCl, then removed and placed in physiological saline (4 °C). Coronal slices were cut and TALs isolated from the outer medulla under a stereomicroscope at 4–10 °C. TALs ranging from 0.7 to 1.0 mm were transferred to a temperature-regulated chamber and perfused using concentric glass pipettes at 37 1 °C. Luminal perfusion rates were 0 or 20 nL min1, and basolateral flow rate was 1 mL min1.
Animals
Measurements of Cai
Male Sprague-Dawley rats weighing 100–150 g from Charles River Breeding Laboratories, Wilmington, MA, were fed a diet containing 0.22% sodium and 1.1% potassium (Purina, Richmond, IN, USA) for at least 5 days prior to the experiments. All protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Case Western Reserve University following the National Institutes of Health Guidelines for Use and Care of Experimental Animals.
Isolated TALs were loaded with 1 lmol L1 FURA 2AM for 30 min and washed for 30 min using dye-free physiological saline at 37 1 °C. The TRPV4 antagonists (ruthenium red and RN1734) and the ATP scavenger hexokinase were present in both luminal and basolateral sides throughout the 30 min-washing period. FURA 2 was excited at 340 nm and 380 nm. The ratio between the emitted fluorescence of both wavelengths is proportional to Cai. Fluorescence was imaged digitally with an image intensifier and a
320
© 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12528
P D Cabral et al.
charge-coupled device camera. Fluorescence images were taken every 2-s in the absence and presence of luminal flow. Data were recorded using METAFLUOR version 7 imaging software (Universal Imaging, Downington, PA, USA). Cai was calibrated at the end of each experiment with 5 mmol L1 EGTA and 10 lmol L1 4-Br-A23187. Cai was calculated using the following equation as previously described: ½Cai ¼ Kd ðFmin =Fmax Þ ðR Rmin Þ=ðRmax RÞ where Kd is the Ca dissociation constant of FURA 2 (224 nmol L1); R is the ratio between 340 nm and 380 nm of each sample; Fmin is the fluorescence intensity of 380 nm during EGTA; Fmax is the fluorescence intensity of 380 nm during 4-Br-A23187; Rmin is the minimum ratio between 340 nm and 380 nm during EGTA, and Rmax is the maximum ratio between 340 nm and 380 nm during 4-Br-A23187. For each experiment where luminal flow was tested, we calculated and analysed the following variables: peak increase of Cai (nmol L1); rate of rise (nmol L1 s1); half-life of the decay (s); and the plateau of the decay (nmol L1). In the set of experiments where the TRPV4 agonist GSK1016790A was tested in the absence of luminal flow, the peak increase of Cai and the rate of rise were analysed.
In vivo adenoviral transduction of medullary thick ascending limbs Small hairpin TRPV4-RNA (shRNA)-expressing adenoviruses under the control of the H1 mouse RNA polymerase promoter were delivered to mTAL as previously described (Ortiz et al. 2003, Silva & Garvin 2008). Briefly, rats were anesthetized with ketamine (60 mg kg1 body wt i.p.) and xylazine (20 mg kg1 body wt i.p.). After anaesthesia, the left kidney was exposed through a flanked incision and the renal artery and vein clamped. Then, four infusions of 20 lL of adenovirus at a final concentration of 1.2 9 1012 particles per mL were injected at the corticomedullary boundary at a flow rate of 20 lL min1 using a 30-gauge needle and syringe pump. The needle was removed from the injection site 30 s after each infusion was completed to avoid bleeding and leakage of the virus. After a total time of 8 min, the vascular clamps were removed, the kidney returned to the abdomen and the wound sutured. After surgery, animals were kept warm and under direct observation until they recovered from the anaesthesia and appeared alert. Then, they were returned to their cages, where food and water were available ad libitum. Experiments were performed 72 h after surgery.
· TRPV4 mediates flow-induced TAL Cai
2+
increase
Statistical analysis Results are presented as mean standard error. An unpaired Student’s t-test was used to calculate the difference between no flow and flow conditions in separated experimental groups. A P value of
