The calcium sensing receptor modulates fluid reabsorption and acid secretion in the proximal tubule

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& 2013 International Society of Nephrology

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The calcium sensing receptor modulates fluid reabsorption and acid secretion in the proximal tubule Giovambattista Capasso1, Peter J. Geibel2, Sara Damiano1, Philippe Jaeger3, William G. Richards4 and John P. Geibel2,5 1

Department of Nephrology, Second University of Naples, Naples, Italy; 2Department of Surgery, Yale University School of Medicine, New Haven, Connecticut, USA; 3Centre for Nephrology, Royal Free Hospital, University College of London, London, UK; 4Amgen Inc., Thousand Oaks, California, USA and 5Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut, USA

The proximal tubule uses a complex process of apical acid secretion and basolateral bicarbonate absorption to regulate both luminal acidification and fluid absorption. One of the primary regulators of apical acid secretion is the luminal sodium–hydrogen exchanger expressed along the apical membrane of the proximal tubule. Similarly, the calciumsensing receptor (CaSR) is also located along the luminal membrane of the proximal tubule. Here we investigated the role of CaSR in proton secretion and fluid reabsorption in proximal tubules by modulating luminal calcium concentration, using both in vivo micropuncture in rats and in vitro perfused mouse proximal tubules. Using CaSR knockout mice and a calcimimetic agent, we found that increased proton secretion and fluid reabsorption were CaSR dependent. Activating CaSR by either raising the luminal calcium ion concentration or by the calcimimetic caused a concomitant increase in sodium-dependent proton extrusion and fluid reabsorption, whereas in proximal tubules isolated from CaSR knockout mice varying calcium ion concentration had no effect. Application of a calcimimetic in lower concentrations of calcium ion stimulated these processes in vitro and in vivo. Thus, in both rats and mice, increased luminal calcium concentration leads to enhanced fluid reabsorption in the proximal tubule, a process related to activation of CaSR. Kidney International (2013) 84, 277–284; doi:10.1038/ki.2013.137; published online 24 April 2013 KEYWORDS: calcium-sensing receptor; intracellular pH; ion transport; kidney stones; kidney tubule

Correspondence: John Geibel, Departments of Surgery, and Cellular and Molecular Physiology, Yale University School of Medicine, BML 238, 310 Cedar Street, New Haven, Connecticut 06520, USA. E-mail: [email protected] Received 9 August 2012; revised 5 February 2013; accepted 7 February 2013; published online 24 April 2013 Kidney International (2013) 84, 277–284

Acid secretion in the proximal tubule (PT) is a complex process that involves both basolateral and apical proteins, which balance the excretion of hydrogen ions with the absorption of bicarbonate. The PT is responsible for 60–80%1–4 of all bicarbonate reabsorption; this reabsorption is modulated by hormonal and environmental causes, leading to facilitated and active insertion of ion transport proteins on both the apical and basolateral poles of the proximal tubular cell. Increased bicarbonate absorption requires enhanced proton secretion at the apical membrane, by activation of apical sodium–hydrogen exchanger (NHE) and H-ATPase,5–9 and the concomitant upregulation of the sodium bicarbonate cotransporter protein on the basolateral membrane.10 Previously, our group has shown that exposure to angiotensin II upregulates the NHE and the sodium bicarbonate cotransporterproteins, leading to enhanced fluid uptake and HCO3 reabsorption.1 These studies were followed by a series of articles demonstrating that angiotensin II can also regulate the insertion of the V-type ATPase into the apical membrane of the cell, inducing enhanced proton extrusion into the lumen.11 This trafficking to the surface is linked to a functional Cl channel in the vesicles containing the V-ATPase.12–14 The calcium-sensing receptor (CaSR) is a member of the pheromone class of G-protein-coupled receptors15 that is expressed in a variety of tissues throughout the body and has been identified to have a number of physiological effects, including modulation of fluid and electrolyte transport, regulation of calcium uptake, modulation of bone formation, as well as cell proliferation and differentiation.16–19 In the parathyroid gland it is responsible for regulating body calcium homeostasis by modulating the levels of parathyroid hormone and calcium.20 In the stomach it stimulates acid secretion,21–23 whereas in the intestine it helps regulate fluid and electrolyte absorption and secretion by sensing the concentrations of electrolytes, amino acids, and polyamines.22,24 Studies by Riccardi et al.25–28 demonstrated that the CaSR is found throughout the kidney and that it is expressed on the apical surface of the PT. 277

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G Capasso et al.: CaSR modulates fluid reabsorption and acid secretion

In the present study we examined the role of CaSR in modulating acid secretion and fluid absorption (Jv) in the PT. To this end, we used a combination of in vivo (micropuncture) and in vitro techniques (isolated perfused tubules) from rats and mice, respectively, to examine the local effects of luminal Ca2 þ concentration on both tubular fluid reabsorption and acid–base regulation in the PT. In parallel studies we demonstrate that activation of the receptor by a calcimimetic leads to identical effects on both tubular fluid volume and acid secretion at low levels of calcium. These studies add additional evidence that by allosteric- or chemical-induced modulation of the receptor, we can alter cellular parameters by slight modulations in luminal calcium concentration.

control Jv 0.337±0.004 to 0.142±0.018 or 0.206±0.009, respectively.

RESULTS In vitro perfusion studies Dose-dependent changes in Jv, while modulating luminal Ca2 þ concentrations in rats. In this series of studies, rat PTs

S2 segments from cortical nephrons were perfused with various luminal Ca2 þ concentrations from 0.1 to 10 mmol/l, while maintaining a constant bath Ca2 þ concentration of 1.2 mmol/l (Figure 1). The data shown are from five different rats for each data point, in which the concentrations of the apical solutions were randomly changed; there was a 5-min period between each collection where the collected perfusate was discarded to prevent contamination from the previous sample. We were able to demonstrate that increasing luminal Ca2 þ concentrations gave a highly significant Po0.0001 change in the rate of Jv in nl/mm/min. We were able to show that reducing the Ca2 þ concentration to 0.1 mmol/l or 0.5 mmol/l resulted in a significant reduction from the

(Casr þ / þ ::Gcm2 þ / þ ) mice to determine whether modification in the delivered Ca2 þ concentration to the apical surface of the PT affects the rate of fluid absorption in this segment of the nephron. Figure 2 demonstrates that changes in the luminal concentration of Ca2 þ results in a significant modulation of the rate of fluid reabsorption in a dosedependent manner in Casr þ / þ ::Gcm2 þ / þ mice. At low Ca2 þ concentration (0.1 mmol/l), Jv is restricted to 0.103±0.009 when compared with physiological Ca levels (1.2 mmol/l) 0.334±0.005. By approximately doubling the initial luminal Ca2 þ concentration (1.2–2.0 mmol/l), we observed a further significant increase in tubular fluid reabsorption up to 0.576±0.017 (Po0.0001; see Figure 2). These data were comparable to the data found in the rat studies, suggesting similar modes of action of the receptor across species. For these studies we used isolated tubules from five mice with three tubules per mouse. In a separate series of studies we examined whether the changes in the rate of fluid absorption required a functional CaSR on the apical membrane of the PT by repeating the experiment in PTs isolated from Casr  /  ::Gcm2  /  mice. Following PT isolation and perfusion, we again varied the luminal Ca2 þ calcium concentration from 0.1 to 2.0 mmol/l, while collecting the luminal fluid. Under each period we discarded the first two collections following the change in the P<0.0008 0.8 NS 0.6 nl/mm/min

P<0.0001 P<0.0001 P<0.0001 P<0.0001

0.6

Jv following modifications in luminal Ca2 þ concentration in mice. Individual PTs were isolated from wild-type

P<0.0001 0.4

0.2

P<0.0001 P<0.0001

/l ol m

m

a 2+ C

ol /l

ol /l

C

C ol /l

m m 2

1. 2

m

m

m m 0. 1

C

a 2+

a 2+ C

a 2+

/l

C

ol m

m

m

m

ol 5

10

a 2+

/l

C

a 2+

/l

m

m

ol 4

a 2+

C 2

1

m

m

ol

/l

/l

C 0.

5

m

m

ol

/l ol m

m 1 0.

C

a 2+

v lJ tro on C

Figure 1 | Luminal Ca2 þ dose curve on fluid reabsorption (Jv) in rat proximal tubule. Bath Ca2 þ was maintained at a constant 1.2 mmol/l. Data are from three tubules per concentration, with three collections made for each concentration and five rats per data set. N.S., not significant.

a 2+

v on tro

0.2

0

278

a 2+

0.0 lJ

0.4

C

nl/mm/min

NS

Figure 2 | Increasing luminal Ca2 þ concentration enhances net fluid reabsorption (Jv, nl/mm/min) of proximal tubule in the Casr þ / þ ::Gcm2 þ / þ mouse. All data are the means±s.d. from three sequential collections per tubule, with two tubules per mouse used and five animals used for each group. P-values were calculated using GraphPad and a paired Student’s t-test. Control Jv was a solution that contained 1.2 mmol/l CaCl. This solution did not differ from the control perfusate; 1.2 mmol/l CaCl solutions were identical to the initial control, except that they were added at a different randomized perfusion time point. The order of solution exchanges in the lumen was randomized; to prevent contamination from the previous solution after the exchange, fluid generated during the first 5 min following the switch was collected and discarded. N.S., not significant. Kidney International (2013) 84, 277–284

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G Capasso et al.: CaSR modulates fluid reabsorption and acid secretion

NS

NS

NS

P<0.0001

NS

0.3

NS P<0.0001 P <0.0001

0.2

3 nl/mm/min

nl/mm/min

4

0.1

P<0.0008 2

1 a 2+

apical perfusion, to prevent collection of any mixed perfusate. As shown in Figure 3, changes in the luminal Ca2 þ concentration had no effect on the rate of fluid absorption in the absence of a functional CaSR, 0.247±0.006 (control) versus 0.235±0.012 (2.0 mmol/l), indicating that the receptor acts to modulate the rate of fluid reabsorption based on receptor activation. Of interest were the reduced initial Jv values as compared with mice with a functional receptor (0.247±0.006). We used five Casr  /  ::Gcm2  /  mice and perfused three tubules per mouse to mean the data that is presented for each group. In vivo perfusion studies

In these studies single PTs were perfused with a variety of Ca2 þ concentrations. At each concentration, 12 PTs/rat were perfused from five rats and averaged. Absolute fluid absorption (Jv; expressed as nl/mm/min) measured in the PT averaged 1.15±0.48 in rats perfused with 1.2 mmol/l Ca2 þ . Similar to results obtained from ex vivo perfused PTs, increasing Ca2 þ to 2 mmol/l significantly increased Jv to 2.35±0.52 (Po0.001; Figure 4). Moreover, in concordance with the ex vivo data, proximal tubular Jv was inhibited in tubules perfused with 0.1 mmol/l Ca2 þ (0.62±0.15 nl/mm/min; Po0.001; Figure 4).

tro l on C

2

m

m

ol /l C

a 2+

a 2+ ol /l C m m

m m 1 0.

1

m

m

ol /l C a 2+

ol /l C

a 2+ 2

m 2

Figure 3 | Variations of luminal Ca2 þ concentration in the Casr  /  ::Gcm2  /  mouse and the effects on fluid reabsorption (Jv). Note that lack of a functional receptor prevents Ca2 þ from having an effect on Jv. The data represent three sequential collections under each experimental condition. There were three tubules per mouse, and five mice were used for the study. All data are the means±s.d. from three sequential collections per tubule, with three tubules per mouse used and five animals for each group. P-values were calculated using GraphPad and a paired Student’s t-test. Control Jv was a solution that contained 1.2 mmol/l CaCl. This solution did not differ from the control perfusate. This was the Jv that was recorded following perfusion with either a low (0.1 mmol/l) or high (2.0 mmol/l) CaCl solution. The order of solution exchanges in the lumen was randomized; to prevent contamination from the previous solution after the exchange, fluid generated during the first 5 min following the switch was collected and discarded. N.S., not significant.

Kidney International (2013) 84, 277–284

0

1.

0. 1

m

C

m

m ol /l C

ol /l C

on tro lJ

v

a 2+

0.0

Figure 4 | The effect of changes in luminal Ca2 þ concentration on the rate of fluid absorption in single in vivo microperfused rat proximal tubules. Data shown are pooled from 12 tubules (5 rats). Each tubule was sequentially perfused with the perfusion solutions containing different Ca2 þ concentrations. N.S., not significant.

Effects of calcimimetics perfusion on Jv In vitro perfusion studies. In this series of studies we

investigated whether the calcimimetic agent R-568 (100 nmol/l)24 could modulate fluid absorption in isolated perfused PTs from both wild-type Casr þ / þ ::Gcm2 þ / þ and CaSR-deficient Casr  /  ::Gcm2  /  mice. We used the same tubule isolation and perfusion techniques described above, with the only difference being the inclusion of R-568 to the luminal perfusate for some studies. As shown in Figure 5, addition of R-568 (100 nmol/l) to tubules with 0.1 mmol/l luminal Ca2 þ concentration resulted in a Jv rate (0.334±0.016) comparable to control perfusions with 1.2 mmol/l Ca2 þ (0.332±0.006). Figure 6 demonstrates that the effects of R-568 on Jv are selective for CaSR, as Jv of mice lacking the receptor cannot be stimulated by adding R-568 to the perfusate (0.231±0.006 vs. 0.233±0.012). The rates of perfusion in low Ca2 þ ±R-568 remain low in CaSR knockout mice compared with controls that have a functional receptor. In vivo studies. In a separate series of studies we examined whether the calcimimetic agent R-568 (100 nmol/l) could stimulate fluid absorption in the intact nephron. As shown in Figure 7, perfusion of the PT with R-568 and low luminal Ca2 þ (0.1 mmol/l) resulted in an increase in tubular fluid absorption (2.22±0.57 vs. 0.62±0.15) that was comparable to perfusing the lumen with high (2.0 mmol/l) Ca2 þ alone (2.35±0.52; see above and in Figure 4). These data confirm that treatment with a calcimimetic agent increases the sensitivity of the CaSR to Ca2 þ so that low concentrations of extracellular Ca2 þ can lead to receptor activation, resulting in a significant physiological response. 279

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NS

P<0.0005

P<0.0001

P<0.0001 P<0.0004

P<0.0001

0.4

2.5 2.0 nl/mm/min

nl/mm/min

0.3 0.2

1.5 1.0

0.1 0.5 0.0

C a 2+ /l m m ol

ol /l m

m

1

1. 2

m

m 1

0.

0.

0.

Figure 5 | Effects of calcium-sensing receptor activator R-568 on Jv in Casr þ / þ ::Gcm2 þ / þ mice perfused in vitro. Please note that addition of R-568 to the luminal perfusate in the continued presence of low Ca2 þ increased fluid reabsoprtion (Jv) to levels comparable to control. N.S., not significant.

C R a 2+ -5 + 68

C a 2+ ol /l

1 m 10 mo 0 l/l nM C R a 2+ -5 + 68

ol /l C a 2+ m m 1 0.

1.

2

m Co m nt ol ro /l l C Jv a 2+

0

Figure 7 | Effect of calcium-sensing receptor activator R-568 (100 nmol/l) on absolute fluid absorption (Jv) in proximal tubule of rats. The addition of R-568 to the luminal perfusate increased Jv to values comparable to those obtained with 2 mmol/l Ca2 þ in the perfusion solution. N.S., not significant.

NS NS NS NS P<0.0001

NS

0.3

P<0.0001 NS

0.08

0.2

NHE3 ΔpHi /Δt

nl/mm/min

0.10

0.1

0.06 0.04 0.02

R 56 8 nM

co

/–

aS

R–

/–

+4 00

C aS R–

W T+

40 0

nM

R

nt ro

l

-5 68

tro l co n

C

Figure 6 | Effects of the calcium-sensing receptor activator R-568 on fluid reabsorption (Jv) in Casr  /  ::Gcm2  /  mice perfused in vitro. Note that addition of R-568 to the luminal perfusate in the continued presence of low Ca2 þ did not modify Jv from initial low levels. N.S., not significant.

0

W T

1 m 10 mo 0 l/l nM C R a 2+ -5 + 68

0.

a 2+ C ol /l m m 1 0.

1. 2

m Co m nt ol ro /l l C Jv a 2+

0

Figure 8 | Effects of luminal perfusion of R-568 on Na þ dependent acid extrusion from Casr þ / þ ::Gcm2 þ / þ and Casr  /  ::Gcm2  /  mice. Addition of 400 nmol/l of the calcimimetic R-568 leads to a significant increase in Na þ -dependent proton extrusion from mouse proximal tubules. The lack of a functional calcium-sensing receptor (CaSR) prevents an enhanced sodium–hydrogen exchanger (NHE) activity in the tubule.

Role of R-568 on NHE in the PT

To determine what effects R-568 may have on PT acidification, and specifically on the apical NHE, which we previously demonstrated that in the colon is modulated by exposure to 280

calcimimetics,24,29 we conducted studies in isolated perfused PTs from either Casr þ / þ ::Gcm2 þ / þ or Casr  /  ::Gcm2  /  mice. Figure 8 provides a summary of five mice and three tubules Kidney International (2013) 84, 277–284

G Capasso et al.: CaSR modulates fluid reabsorption and acid secretion

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from each mouse, in which measurements of intracellular pH were performed in the isolated perfused PT looking at the Na þ -dependent pH recovery from an acid load, a direct measure of NHE activity, in the presence or absence of 400 nmol/l luminal R-568 at a constant Ca2 þ concentration (1.2 mmol/l). Addition of R-568 to the perfusate led to a rapid increase in the rate of Na þ -dependent H þ -ion extrusion from the cell (dpH/dT 0.071±0.007). In CaSRdeficient mice, there was no change in the rate of Na þ -dependent H þ -ion extrusion (dpH/dT 0.037±0.002 vs. 0.0351±0.002), suggesting that the effect of the calcimimetic agent is directly linked to a functional CaSR receptor.

increase the amount of H þ ion secretion in the PT so that Ca2 þ in the lumen of the tubule would become ionized, making it ready for absorption in the more distal portions of the nephron. However, we cannot rule out from the present studies that receptor activation and the concurrent NHE stimulation may lead to enhanced absorption through the paracellular pathway in the PT. When we modulated the Ca2 þ concentration in the PT, either in vivo using a rat micropuncture technique2 or in vitro with the isolated perfused tubule,35,36 we observed the following effects as compared with the physiological luminal Ca2 þ concentration (1.2 mmol/l); the reduction of Ca2 þ in the lumen perfusate (0.1 mmol/l) reduced Jv, whereas the opposite was observed by increasing luminal Ca2 þ (2.0 mmol/l and beyond; see Figures 1–4). Utilizing perfused PTs from the rat (Figure 7) or from the Casr þ / þ :: Gcm2 þ / þ mice (Figure 2) we demonstrated that the Ca2 þ dependent modulation in fluid reabsorption in the PT was CaSR dependent, as the specific calcimimetic agent R-568 enhanced transepithelial flux in the presence low Ca2 þ . Furthermore, in perfused PTs isolated from Casr  /  ::Gcm2  /  mice, no change in Jv was observed in response to Ca2 þ either alone or in the presence of R-568, in either low (0.1 mmol/l) or high (2.0 mmol/l) luminal Ca2 þ (see Figure 3). These data indicate that a functional CaSR is required for the tubule to modulate transepithelial fluid flux in response to changes in apical divalent ion concentration. Of interest is that there is a reduction in Jv in animals lacking expression of the CaSR when compared with wild-type animals, suggesting that lack of a functional receptor leads to reduced fluid absorption along the nephron. Following our observation that stimulation of the receptor leads to an enhancement in fluid absorption, we wanted to see whether the receptor modulates NHE activity in the renal tubules as it does in the colon.24,34 We used an isolated perfused PT preparation to investigate the apical activation of the receptor on NHE activity by directly tracking changes in intracellular pH with a fluorescent intracellular pH indicator BCECF (20 ,70 -bis(2-carboxyethyl)-5(6)-carboxyfluorescein) and a high-speed transfer charge-coupled device camera as we had previously described.37 When the tubules were exposed to a calcimimetic agent we observed a significant increase in sodium-dependent H þ extrusion via NHE. We calculated the rate of proton efflux at the same initial pH so that intrinsic buffering was constant for all pH calculations and correcting for any changes in endogenous buffering power as previously described.1,10 By using this method we demonstrated that activation of the CasR in the PT of wildtype animals by a calcimimetic agent leads to direct activation of the NHE protein in that segment of the nephron (Figure 8). These observations coupled with the data on transepithelial flux in response to CaSR activation suggest a model in which receptor activation leads to enhanced H þ extrusion and, consequently, Na þ reabsorption that ultimately will explain the observed increase in fluid reabsorption. On the other hand, activation of NHE will

DISCUSSION

Following the cloning of the CaSR, studies were conducted to determine the expression of the receptor in a variety of tissues. In the elegant studies by Riccardi et al.,30 the receptor was identified in the kidney and was specifically shown on the apical surface of the PT. Owing to the high selectivity for Ca2 þ , one could speculate that the receptor was sensing the amount of delivered Ca2 þ to the PT, and that depending on the concentration could increase or decrease Jv to allow for a reduction in the accumulation of Ca2 þ along the remainder of the nephron. In addition to delivered Ca2 þ it should be pointed out that the receptor also senses salinity and intraluminal pH,20,31,32 so that the receptor is acting as a sensor of the intraluminal environment in the PT and would modulate fluid reabsorption along with Ca2 þ concentration. As shown in this study, modulations in Ca2 þ will also regulate apical NHE activity and could thereby add more acid to the lumen, which would lead to additional ionized Ca2 þ that could be absorbed in more distal segments of the nephron. A recent study by Loupy et al.33 using commercial antibodies against specific fragments of the receptor suggested a different profile of receptor localization and density within the nephron from that previously reported. This recent result appears contradictory to our in vivo and in vitro functional studies; however, it is well known that because of the complex architecture of the receptor it is often difficult to obtain staining and localization of the receptor. Riccardi et al.20,25,30 used polyclonal antibodies against both full-length CaSR and specific regions of the exofacial domains coupled with antigen retrieval to obtain elegant images of the receptor localized to the apical surface of the PT. When you take our in vivo and in vitro data from two separate species where we demonstrate an effect of modulations in the Ca2 þ concentration on fluid movement, and further show this is specific to a calcimimetic agent, we feel confident that these data represent the functional role of the receptor in the PT. Furthermore, in vitro data generated from CaSR-deplete animals confirm the specific actions of the CaSR in the PT to modulate both fluid reabsorption and acid secretion. We further postulated that the receptor, which our group has previously shown to interact with the NHE3 protein in the colon,24,34 may have a similar effect in the kidney, namely to Kidney International (2013) 84, 277–284

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favor the ionization of calcium so that the ionized calcium is delivered to the distal portions of the nephron where it would be more easily reabsorbed, thus avoiding an increase in the luminal Ca2 þ concentration. Therefore, we speculate that the luminal CaSR along the PT acts as a modulator of both fluid and Ca2 þ absorption, thus making it a key element to avoid Ca2 þ precipitation along more distal segments of the nephron. In conclusion, we have demonstrated that luminal CaSR has an active role in modulating PT fluid absorption as well as acid secretion, and we envision its possible involvement in prevention of renal stone disease.

Table 1 | Composition of solutions in in vitro studies

MATERIALS AND METHODS In vitro–isolated tubule perfusion Rats and mice. Overnight fasted male Sprague–Dawley rats (120–150 g) and Casr þ / þ ::Gcm2 þ / þ or Casr  /  ::Gcm2  /  mice were used in accordance with the humane practices of animal care established by the Yale Animal Care and Use Committee. The generation of Casr  /  ::Gcm2  /  mice has been previously described.38 To obtain isolated PTs for perfusion, measurement of cell pH or Jv, male Sprague–Dawley rats 120–150 g and 20–25 g Casr þ / þ ::Gcm2 þ / þ or Casr  /  ::Gcm2  /  mice were anesthetized with isoflurane (Butler Animal Health Supply, Dublin, OH) before excision of the kidney. Following excision, the kidney capsule was removed and the kidney was cut into 5-mm longitudinal sections as previously described.39 PT perfusion and Jv measurement. Single, hand-dissected PTs were placed in a temperature-controlled chamber on the stage of an inverted microscope and perfused in vitro as previously described.1,10,40 Briefly, PTs were perfused (4–8 nl/min) with a solution containing exhaustively dialyzed fluorescein isothiocyanate– inulin (Sigma, St Louis, MO).29,41–43 The effluent was sampled with a volume-calibrated pipette. netJv (nl/min per mm of tubule length) was determined from the length and diameter of the PT, the rate at which the effluent accumulates in the collection pipette, and from the relative fluorescence activity of fluorescein isothiocyanate–inulin in the perfusate and effluent, following the technique previously described.29,41,44 A sample of bath solutions was also collected for each experiment; data were discarded whenever the bath (fluorescein isothiocyanate–inulin) exceeded the background. Each data point consisted of the average of three 5-min collections of effluent. At least five PTs from at least three animals were studied in each experimental protocol. PT viability was assessed by trypan blue exclusion and lack of fluorescein isothiocyanate accumulation in the bath. All secretagogues were added to the luminal perfusate. CaSR modulators were added to the luminal perfusates as indicated. The composition of these solutions can be found in Table 1. Intracellular pH measurements. Isolated individual PTs were transferred to the stage of an inverted microscope and were cannulated using a series of fabricated concentric glass micropipettes, and were perfused in vitro.10,35 All subsequent solution changes, either apical or basolateral, were performed on the tubules following cannulation and perfusion. Measurements of intracellular pH as a means of determining net proton extrusion were conducted by loading PTs with the cellpermeant pH-sensitive dye 20 ,70 -bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM, Invitrogen, Eugene, OR) for 15 min at 37 1C, with a final concentration of 10 mmol/l in 1 ml of HEPES 282

NaCl NMDG NH4Cl KCl MgSO4 CaCl2 Glucose HEPES pH (37 1C)

Solution 1

Solution 2

Solution 3

High K þ calibration

125 — — 3 1.2 1 5 32.2 7.4

— 125 — 3 1.2 1 5 32.2 7.4

— 125 20 3 1.2 1 5 32.2 7.4

— 125 — 105 1.2 1 — 32.2 7.0

All solutions were pH adjusted to 7.4, except high K þ calibration to 7.0. For all solutions, osmolality was adjusted to 300 mOsm. All concentration units are in mmol/l.

Ringer solution (see Table 1, Solution 1). Following loading, the tubules were perfused with a HEPES Ringer solution for 5 min at 37 1C, to remove any de-esterified dye on the basolateral or apical membrane of the tubule. Following dye loading, tubules were sequentially excited with 490 and 440 nm light, while measuring the emission at 535 nm. This ratiometric data was recorded to the computer along with the individual images from the emission spectra using a technique that has been described in detail previously.10,11 These final ratiometric data were converted to pHi following exposure to a Nigericin calibration solution.10,11 To compensate for potential changes in intracellular buffering power, rates of acidification were calculated from the same initial pH following a protocol that was previously used.11 In vivo perfusion studies Rats. Animal preparation. Male Sprague–Dawley rats (200–220 g) were anesthetized with thiobarbital (Inactin, 80 mg/kg intraperitoneally; Research Biochemicals, Natick, MA) and were prepared for in vivo micropuncture studies. Cannulae were placed in a jugular vein for infusion of fluids and in a femoral artery for the recording of mean arterial pressure (Powerlab, AD Instruments, Colorado Springs, CO). A tracheotomy tube was inserted. Animals were allowed to breath room air spontaneously. A catheter was inserted in the left ureter to collect urine. The left kidney was exposed by a flank incision and was stabilized in a Lucite cup mounted on a heated surgical table, and was bathed in mineral oil maintained at 37 1C. After surgical preparation, rats were infused with a solution of 0.154 mol/l of NaCl to maintain euvolemia. Studies were begun after 60 min of stabilization. Microperfusion of PT. The PT site was identified by injections from a ‘finding’ pipette containing dye-stained artificial tubular fluid. The flow was blocked by injection of T grease (T grade, Apiezon Products, Manchester, UK) via a micropipette (10- to 12-mm optical density) proximal to the perfusion site. The tubule was perfused with a micropipette (8- to 10-mm optical density) connected to a microperfusion pump at 18±3 nl/min. The perfusion solution contained 14C-inulin (New England Nuclear, Wellesley, MA) as a volume marker and 0.1% FD&C green dye for identification of the perfused loops. Tubules were perfused for 2–8 min before fluid collections, which were made at a downstream site with a micropipette (7- to 10-mm optical density) after placement of a column of oil to block downstream flow. Samples were collected for 3 to 5 min and were transferred into a constant-bore capillary tube to measure the tubular fluid volume. Thereafter, the samples were injected into scintillation fluid and Kidney International (2013) 84, 277–284

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G Capasso et al.: CaSR modulates fluid reabsorption and acid secretion

Table 2 | Composition of solutions for studies with various Ca2 þ concentrations

REFERENCES 1.

Calcium (mmol/l)

NaCl NMDG NH4Cl KCl MgSO4 CaCl2 Glucose HEPES pH (37 1C)

0.1

1.2

2.0

4.0

5.0

10.0

125.9 — — 5 1.2 1 5 32.2 7.4

124.8 — — 5 1.2 1.2 5 32.2 7.4

123 — — 5 1.2 2.0 5 32.2 7.4

122 — — 5 1.2 4.0 5 32.2 7.4

121

116

5 1.2 5.0 5 32.2 7.4

1.2 10.0 5 32.2 7.4

2.

3. 4. 5.

6.

All solutions were pH adjusted to 7.4 and osmolality was adjusted to 300 mOsm. All concentrations are in mmol/l.

7.

Table 3 | Composition of solutions in vivo studies

8.

Protocol 1

Protocol 2

Protocol 3

Protocol 4

125 20 1 5 1.0 2 5 4.0 7.4

125 20 1 5 1.0 1.2 5 4 7.4

125 20 1 5 1.0 0.1 5 4 7.4

125 20 1 5 1.0 0.1 5 4 7.4

9.

NaCl NaHCO3 NaHPO4 KCl MgSO4 CaCl2 Glucose Urea pH (37 1C)

All solutions were pH adjusted to 7.4, except high K þ calibration to 7, and osmolality was adjusted to 300 mOsm. For Protocol 4, 100 nmol/l R-568 was added to the solution from a 1 mmol/l dimethyl sulfoxide stock solution. All concentrations are in mmol/l.

10.

11.

12.

13.

14. 14 C radioactivity measured. Collected samples with p90% and X105% of the microperfused inulin were discarded. The amount of microperfused inulin was estimated by the average of 14C-activity in four samples microperfused directly into capillary bore tubes at the end of the experiment. To determine the lengths of the perfused segments, tubules were filled with high-viscosity microfil (Flow Tech, Kalamazoo, MI). At the end of the experiments, the kidney was partially digested in 20% NaOH, and the casts were measured under a dissecting microscope. Jv was calculated by the difference between the perfusion rate and the collection rate factored by the length of the nephron: Jv ¼ Vperf (nl/min)  Vcoll (nl/min)/PT length (mm), where Vperf indicates perfusion rate and Vcoll indicates collection rate, and is expressed as nl/mm/min.45 The composition of the perfusion fluid is described in Tables 2 and 3.

Statistics All statistics performed on the data were carried out using a Students paired t-test using the program Prism GraphPad (La Jolla, CA).

15.

16.

17.

18. 19.

20.

21.

22. 23.

DISCLOSURE

J.G. and G.C. received support for this research from Amgen. W.G.R. is an employee of Amgen. All the other authors declared no competing interests.

24.

25.

ACKNOWLEDGMENTS

This work was supported in part by Amgen. Kidney International (2013) 84, 277–284

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Kidney International (2013) 84, 277–284