Effects of angiotensin II on the CO2 dependence of HCO3– reabsorption by the rabbit S2 renal proximal tubule
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《美国生理学杂志》
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut
ABSTRACT
Previous authors showed that, at low doses, both basolateral and luminal ANG II increase the proximal tubule's HCO3– reabsorption rate (JHCO3). Using out-of-equilibrium CO2/HCO3– solutions, we demonstrated that basolateral CO2 increases JHCO3. Here, we examine interactions between ANG II and CO2 in isolated, perfused rabbit S2 segments. We first used equilibrated 5% CO2/22 mM HCO3–/pH 7.40 in bath and lumen. At 10–11 M, basolateral (BL) ANG II increased JHCO3 by 41%, and luminal ANG II increased JHCO3 by 35%. At 10–9 M, basolateral ANG II decreased JHCO3 by 43%, whereas luminal ANG II was without effect. Second, we varied [CO2]BL from 0 to 20% at fixed [HCO3–]BL and pHBL. Fractional stimulation produced by BL 10–11 M ANG II falls when [CO2]BL exceeds 5%. Fractional inhibition produced by BL 10–9 M ANG II tends to rise when [CO2]BL exceeds 5%. Regarding luminal ANG II, fractional stimulation produced by 10–11 M ANG II fell monotonically as [CO2]BL rose from 0 to 20%. Fractional inhibition produced by 10–9 M ANG II rose monotonically with increasing [CO2]BL. Viewed differently, ANG II at 10–11 M tended to reduce stimulation by CO2, and at 10–9 M, produced an even greater reduction. In conclusion, the mutual effects of 1) ANG II on the JHCO3 response to basolateral CO2 and 2) basolateral CO2 on the JHCO3 responses to ANG II suggest that the signal-transduction pathways for ANG II and basolateral CO2 intersect or merge.
kidney; out-of-equilibrium solutions; acid-base; volume reabsorption
ONE OF THE MAJOR tasks of the kidneys is to participate, along with the lungs, in the regulation of the acid-base balance of the extracellular fluid. For example, it has long been appreciated that acute respiratory acidosis (i.e., a rise in PCO2 that causes a fall in pH) rapidly stimulates renal H+ secretion (8, 16). The proximal tubule (PT) plays a key role in this acid secretion. In addition to reabsorbing a near-isosmotic fluid that represents about two-thirds of the fluid filtered by the glomerulus, the PT reabsorbs 80% of the filtered HCO3– as follows. The PT cell actively secretes H+ into the tubule lumen (1, 6, 35) and uses this H+ to titrate filtered HCO3– in the lumen to CO2 and H2O, catalyzed by apical carbonic anhydrase IV (9, 36, 37). The newly formed CO2 and H2O then diffuse into the PT cells, where carbonic anhydrase II (36, 37) catalyzes the regeneration of H+ and HCO3–. The cell extrudes the H+ across the apical membrane via Na-H exchangers (4, 5, 30) and H+ pumps (20), while exporting the HCO3– across the basolateral membrane, mainly via the electrogenic Na/HCO3 cotransporter (7, 15, 32, 33).
Using out-of-equilibrium (OOE) CO2/HCO3– solutions to alter [CO2], [HCO3–], or [H+] one at a time (40, 41, 43), our laboratory demonstrated that, at least in regard to acute acid-base disturbances, H+ secretion by the PT responds not to changes in pH, but only to changes in basolateral [CO2] and [HCO3–] (43). Thus, in the case of acute respiratory acidosis, the PT cell senses the increase in blood [CO2] per se, which is a powerful stimulus for HCO3– reabsorption.
Perhaps the most powerful hormonal stimulus for HCO3– reabsorption is ANG II. The first report of the effects of ANG II on PT transport was in 1968 by Burg and Orloff (12) on isolated, perfused rabbit PTs. They noted that adding 2 x 10–6 M ANG II to the basolateral solution had no detectable effect on the rate of fluid absorption (JV). In a 1974 rat micropuncture study, Steven (38) reported that 2 x 10–5 M basolateral ANG II lowered JV, whereas 1 x 10–7 M had no effect. Harris and Young (23) later showed that basolateral ANG II has a biphasic effect on Na+ reabsorption in the rat PT, stimulating at low doses (10–12-10–10 M) and inhibiting at higher doses (3 x 10–7-3 x 10–6 M). Shuster and colleagues (34) in 1984 demonstrated a similar biphasic effect of basolateral ANG II on JV in isolated, perfused rabbit PTs, ruling out a role of sympathetic innervation on the JV response. In 1991, working in isolated, perfused rat proximal straight tubules (PSTs), Garvin (19) found that 10–10 M basolateral ANG II increases both JHCO3 and JV. At about the same time, Chatsudthipong and Chan (14) found that high levels of basolateral ANG II reduce JHCO3 in rats and that these effects are blocked by saralasin, an antagonist of ANG II receptors.
As far as the effects of luminal ANG II are concerned, in a 1988 micropuncture study, Liu and Cogan (26) showed that low-dose luminal ANG II (10–12-10–11 M) increases HCO3– reabsorption (JHCO3) even in a denervated rat kidney. The 1990 study on the rat PT by Wang and Chan (39) extended the earlier observations by showing that increasing levels of luminal ANG II have biphasic effects on JHCO3 as well as JV. Consistent with these last two papers, Morduchowicz et al. (29), working in brush-border membrane vesicles, showed that ANG II stimulates Na-H exchange. Li et al. (25) in 1994 and Du et al. (17) in 2003 extended the biphasic effects of ANG II on JV to the lumen of the isolated, perfused rabbit PT. In 1997, working in isolated perfused rabbit proximal convoluted tubule (PCTs), Baum et al. (2) found that, in the presence but not in the absence of a luminal ACE inhibitor, low-dose luminal ANG II increased both JV and JHCO3.
The pattern that emerges from the above work is that, whether applied to the luminal or basolateral surface of the PT, low-dose ANG II generally increases JV and JHCO3, whereas high-dose ANG II generally has the opposite effect. In fact, until our observation that basolateral CO2 is also a powerful stimulus for HCO3– reabsorption (43), low-dose ANG II was the single most powerful known stimulus. The purposes of the present study were to determine whether 1) the effects of basolateral CO2 and low-dose ANG II, added to either the bath (i.e., basolateral solution) or lumen (i.e., luminal solution), are additive and 2) high-dose ANG II antagonizes the stimulatory effects of CO2.
Our approach was to use OOE solutions to vary basolateral [CO2] from 0 to 20% while keeping basolateral [HCO3–] and pH fixed near their physiological values in isolated, perfused rabbit S2 PTs. We found that low-dose (i.e., 10–11 M) ANG II, whether added to the bath or lumen, maximally increased JHCO3 at low basolateral (BL) [CO2], but that the effects tended to wane at high values of [CO2]BL. High-dose ANG II (i.e., 10–9 M) added to the bath produced a uniform decrease in JHCO3, regardless of [CO2]BL. When added to the lumen, high-dose ANG II had no effect when [CO2]BL was 0%, but increasingly larger inhibitor effects at high values of [CO2]BL.
METHODS
Biological preparation. All experiments were carried out in "pathogen-free" female rabbits (New Zealand White, Elite, Covance, Denver, PA) according to procedures that were approved by the Yale Animal Care and Use Committee. We perfused the PTs using methods that were similar to those originally described by Burg et al. (10) and later modified by Baum et al. (2) and also by our laboratory (31, 41). Briefly, a rabbit weighing 1.4–2.0 kg was euthanized by a single overdose of 3 ml (20 mg) of intravenous pentobarbital sodium. An incision of the abdominal wall exposed the left kidney, which we rapidly removed and then cut into coronal slices that we kept in cold (4°C) modified Hanks' solution (solution 1 in Table 1). Microdissection of the slice was carried out by hand in the same solution under a dissection microscope, using fine forceps, to yield individual midcortical S2 segments 1.5–1.7 mm in length, as detailed in Ref. 41. We cannulated the perfusion end of the tubule using concentric holding, perfusion and exchange pipettes. We cannulated the collection end of the tubule using a holding pipette and collected samples using a calibrated collection pipette (volume 55 nl), as summarized in Fig. 1B of Ref. 41. The mean length of perfused tubules in our JHCO3/JV experiments, as measured with an eyepiece micrometer, was 1.22 ± 0.01 mm (n = 99 tubules), representing the distal end of the PCT. The mean luminal collection rate was 12.3 ± 0.1 nl/min (n = 198 collection periods). We perfused the basolateral side of the tubule (i.e., "bath") at 7 ml/min, with a solution at 37°C.
Experimental protocol and solutions. The experimental protocol was similar to that of a previous study (43) except for the addition of ANG II to the luminal or basolateral solutions. Table 1 lists the compositions of the solutions, all of which were identical to those used in the aforementioned study (43). In brief, we dissected PTs in Hanks' solution (solution 1) at 4°C. The luminal perfusate always was solution 2, which contained dialyzed 3H-methoxyinulin (MW 7146, catalogue no. NET-086L, PerkinElmer Life Sciences, Boston, MA) as the volume marker. Solution 3, which contained 2% albumin, flowed through the bath during a 20- to 30-min warm-up period at 37°C. Following the warm-up period, all experiments had two periods for the collection of luminal fluid. During the first of these, the bath contained solution 4, with or without ANG II. During the second collection period, the bath contained ANG II dissolved in solutions 4, 5, 6, 7, or 8. In experiments in which we perfused the lumen with ANG II, the hormone was present in the lumen throughout the experiment; however, in these experiments, ANG II was always absent from the bath during both collection periods. We generated OOE CO2/HCO3– solutions (solutions 5-8) by rapidly mixing streams of two dissimilar solutions (40) and delivering the newly mixed solution to the tubule within 200 ms. All solutions had osmolalities of 300 ± 2 mosmol/kgH2O.
Measurement of JHCO3 and JV. In each of the two collection periods, we allowed the tubule to stabilize in the appropriate bath solution for 5–8 min, removed and discarded the fluid that had accumulated in the holding pipette at the collection end of the tubule, and then began a series of three timed and calibrated collections. The first two were subsequently analyzed for [3H]methoxyinulin for use in the calculation of JV, and the third was analyzed for total CO2 for use in the calculation of JHCO3. Our measurement of JHCO3 (pmol·min–1·mm–1 tubule length) and JV (nl·min–1·mm–1) was similar to that used by McKinney and Burg (28) and identical to our previous approach (41, 43). We determined total CO2 in aliquots of the perfusate and collected fluid using a WPI "NanoFlo" device (World Precision Instruments, Sarasota, FL) together with Diagnostic Kit 132-A (Sigma, St. Louis, MO). In this paper, the values that we report for JHCO3 (or JV) in first collection period are unnormalized mean values. The values that we report for JHCO3 (or JV) in the second collection period are normalized, mean values computed as described previously (43). Briefly, in each experiment, we divided the JHCO3 (or JV) value obtained during the second collection period by the comparable values obtained during the first collection period; the result was a pair of second/first collection-period ratios. We then multiplied 1) the second/first ratio for JHCO3 (or JV) in a particular experiment by 2) the unnormalized, mean, JHCO3 (or JV) value that we obtained during the first collection periods in a series of experiments following the identical protocol.
Data analysis. For comparisons at 0, 5, and 20% basolateral CO2, we performed one-way ANOVA and Dunnett's multiple comparison for ANOVA for five means using KaleidaGraph (Version 4, Synergy Software, Reading, PA). In this analysis, which we applied separately for JHCO3 and JV at each level of [CO2]BL, we compared the control condition (no hormone) with basolateral 10–11 M ANG II, basolateral 10–9 M ANG II, luminal 10–11 M ANG II, and luminal 10–9 M ANG II. For comparisons at 2.5 and 10% basolateral CO2, we performed two-tailed, unpaired t-tests using the Analysis Toolpack of Microsoft Excel. Results are given as means ± SE, with the number of tubules (n) from which it was calculated.
RESULTS
Effects of basolateral ANG II on JHCO3 and JV with an equilibrated CO2/HCO3– solution in the bath. In our first set of studies, we exposed the basolateral surface of S2 PTs to an equilibrated CO2/HCO3– solution, [CO2]BL = 5%, [HCO3–]BL = 22 mM, and pHBL = 7.4 (solution 4), throughout the two collection periods of the experiment. During the first collection period, no hormone was present (Fig. 1A, open bar). During the second collection period, we added to the bath either 10–11 M ANG II (gray bar) or 10–9 M ANG II (filled bar). Thus we were able to compare the effects of ANG II on JHCO3 and JV in the same tubule. Because in our analysis of basolateral ANG II (Fig. 1A) we used the same control data as in our analysis of luminal ANG II (see Fig. 3A), we employed one-way ANOVA for five groups: 1) the identical control data for Figs. 1A and 3A; 2 and 3) the basolateral low- or high-dose ANG II data for Fig. 1A; and 4 and 5) the luminal low- or high-dose ANG II data for Fig. 3A. The overall P value was <0.0001. Dunnett's multiple comparison shows that, compared with the control condition in which no added ANG II was present in the bath, the presence of a "low dose" of 10–11 M ANG II increased JHCO3 significantly from 56 ± 3 to 79 ± 5 pmol·min–1·mm–1 (P < 0.0001), as shown by the first two bars in Fig. 1A. In contrast, adding a "high dose" of 10–9 M ANG II decreased JHCO3 significantly from 56 ± 3 to 33 ± 4 pmol·min–1·mm–1 (P < 0.0001).
Our control mean JHCO3 value of 56 pmol·min–1·mm–1 is similar to the values of 82 pmol·min–1·mm–1 reported by Burg and Green (11) and 62 pmol·min–1·mm–1 reported by Baum et al. (2), both from the isolated, perfused rabbit PCT. The 41% increase in JHCO3 that 10–11 M ANG II produced in our experiments is somewhat higher than the 29% increase that 10–10 M ANG II produced in the experiments in isolated, perfused rat PSTs by Garvin (19).
Figure 1B summarizes the JV data, which we analyzed using the same ANOVA approach summarized in the previous paragraph. The overall P value was 0.00013. The effect of 10–11 M basolateral ANG II on JV was stimulatory, as it was for JHCO3. The low dose of the peptide increased the mean JV from 0.46 ± 0.03 to 0.62 ± 0.05 nl·min–1·mm–1 (P = 0.0087). On the other hand, adding 10–9 M basolateral ANG II did not have a statistically significant effect on JV, which changed from 0.46 ± 0.03 under control conditions to 0.41 ± 0.05 nl·min–1·mm–1 in the presence of the high dose of the peptide (P = 0.8033).
Our control JV value of 0.46 nl·min–1·mm–1 is about two-thirds as great as the value reported by Schuster et al. (34), who worked with a combination of S1 and S2 segments from midcortical and juxtamedullary nephrons. On the other hand, our observed 36% increase of JV with 10–11 M ANG II is about twice as great as that observed by the earlier investigators. Our JV data confirm the earlier observations of others, made in a combination of rat (14, 23, 38) and rabbit (34) PTs, that increasing levels of basolateral ANG II have a biphasic effect on JV. In addition, we observed that increasing levels of basolateral ANG II have a biphasic effect on JHCO3. Our JHCO3 data are consistent with those made by Garvin (19) in rat PSTs for low-dose ANG II, and with those made in rats by Chatsudthipong and Chan (14) for high-dose ANG II.
Effects of basolateral ANG II on the basolateral CO2 dependence of JHCO3 and JV. A previous study from our laboratory demonstrated that increasing [CO2]BL from 0 to 20%, using OOE technology to fix [HCO3–]BL at 22 mM and fix pHBL at 7.4, substantially stimulated bicarbonate reabsorption by the rabbit S2 PT (43). In the present study, we examined the effects of low- or high-dose basolateral ANG II on the basolateral CO2 dependence of JHCO3. In this series of experiments, the bath contained 10–11 or 10–9 M ANG II plus an equilibrated 5% CO2/22 mM HCO3–/pH 7.40 solution during the first collection period, and an OOE solution containing the same level of ANG II and the same 22 mM HCO3–/pH 7.40 but a variable level of CO2 during the second collection period.
Effect of low-dose ANG II. The diamonds in Fig. 2A summarize JHCO3 data obtained in the presence of 10–11 M basolateral ANG II as we increased [CO2]BL from 0% CO2 (solution 5) to 20% CO2 (solution 8) at a fixed [HCO3–]BL of 22 mM and a fixed pHBL of 7.4. The circles summarize comparable control data in the absence of added ANG II. These control data are from an earlier study (43), augmented by 12 additional experiments at 5% CO2 from Fig. 1A. In the previous section, we discussed the use of one-way ANOVA for five groups to analyze the data at a [CO2]BL of 5%. We similarly used one-way ANOVA for five groups (Figs. 2A and 4A) to analyze the data at [CO2]BL values of 0 and 20%. The overall P values were <0.0001 for both the 0 and 20% data. In addition, we used a t-test to analyze the data at [CO2]BL values of 2.5 and 10% in Fig. 2A. Compared with the control condition, 10–11 M basolateral ANG II produced a statistically significant increase in JHCO3 at 0 (P = 0.041) and 5% CO2 (P < 0.0001). The difference was not statistically significant at either 2.5 (P = 0.092) or 10% CO2 (P = 0.51). Basolateral 10–11 M ANG II produced a small but statistically significant decrease in JHCO3 at 20% CO2 (P = 0.032). Viewed differently, low-dose basolateral ANG II steepens relationship between JHCO3 and [CO2]BL at low levels of [CO2]BL (i.e., 0–5%), but eliminated the stimulation by CO2 at higher [CO2]BL levels.
We analyzed the JV data the same way we analyzed the JHCO3 data. For the ANOVA, the overall P values were 0.00088 for 0% CO2 and 0.063 for 20% CO2. Compared with the control situation with no added hormone (circles in Fig. 2B), low-dose basolateral ANG II (diamonds) tended to increase JV, although the effect was statistically significant only at a [CO2]BL value of 5%.
Effect of high-dose ANG II. The squares in Fig. 2A summarize JHCO3 data obtained in the presence of 10–9 M basolateral ANG II as we increased [CO2]BL from 0% CO2 to 20% CO2 at a fixed [HCO3–]BL of 22 mM and a fixed pHBL of 7.4. The statistical analysis of these JHCO3 data was part of the same JHCO3 ANOVA discussed two paragraphs above. Compared with the control condition, high-dose basolateral ANG II produced a statistically significant decrease in JHCO3 at all three levels of [CO2]BL: 0% (P = 0.0049), 5% (P < 0.0001), and 20% (P < 0.0001). Viewed differently, high-dose basolateral ANG II flattens the relationship between JHCO3 and [CO2]BL at low levels of [CO2]BL (i.e., 0–5%) and eliminates CO2 sensitivity at higher [CO2]BL levels.
The statistical analysis of the JV data was part of the same JV ANOVA discussed two paragraphs above. Compared with the control situation with no added hormone (circles in Fig. 2B), high-dose basolateral ANG II (squares) had no significant effect on JV (Fig. 2B).
Effects of luminal ANG II on JHCO3 and JV with equilibrated CO2/HCO3– solutions in the bath. In our next set of studies, all of the data came from the first collection period of experiments. The open bar in Fig. 3A repeats the control JHCO3 data from Fig. 1A. The gray bar in Fig. 3A represents the effect on JHCO3 of perfusing the lumen with 10–11 M ANG II, and the filled bar represents the effect on JHCO3 of perfusing the lumen with 10–9 M ANG II. The statistical analysis of these JHCO3 data was part of the same JHCO3 ANOVA discussed in conjunction with Fig. 1A. Luminal 10–11 M ANG II significantly increased JHCO3 from 56 ± 3 to 76 ± 7 pmol·min–1·mm–1 (P = 0.011). In contrast, adding a "high dose" of 10–9 M ANG II caused JHCO3 to change by a statistically insignificant amount, from 56 ± 3 to 46 ± 4 pmol·min–1·mm–1 (P = 0.34).
The stimulation by 10–11 M ANG II that we observed confirms the observation by others in rat PTs by Wang and Chan (39). These same authors found that 10–8 M luminal ANG II reduced JHCO3 in rat, whereas we observed no significant effect at 10–9 M. In rabbit PCTs, Baum et al. (2) found no effect of luminal ANG II at concentrations from 10–11 to 2 x 10–8 M. However, in the presence of luminal enalaprilat, these authors found that 10–10 M luminal ANG II did indeed increase JHCO3. Two technical differences between our study and that of Baum et al. is that they perfused the bath at 0.5 ml/min (vs. 7.0 ml/min) and added 6 g/dl albumin to the bath throughout the experiment (vs. 2 g/dl only during the warm-up period).
The statistical analysis of the JV data in Fig. 3B was part of the same JV ANOVA discussed in conjunction with Fig. 1B. Luminal 10–11 M ANG II changed the mean JV by a statistically insignificant amount from 0.46 ± 0.03 to 0.60 ± 0.06 nl·min–1·mm–1 (P = 0.14). Others had observed a stimulation by low-dose ANG II on rabbit PTs (17, 25). We found that adding 10–9 M luminal ANG II significantly increased JV from 0.46 ± 0.03 to 0.67 ± 0.06 nl·min–1·mm–1 (P = 0.014), which is in a direction opposite that seen by others in the rabbit (17, 25) or rat (39).
Effects of luminal ANG II on the basolateral CO2 dependence of JHCO3 and JV. In this series of studies, the lumen contained 10–11 or 10–9 M ANG II throughout the experiment. During the first collection period, the bath contained equilibrated 5% CO2/22 mM HCO3–/pH 7.40, while during the second collection period the bath contained an OOE solution with the same 22 mM HCO3–/pH 7.40 but a variable level of CO2.
Effect of low-dose ANG II. The diamonds in Fig. 4A summarize JHCO3 data obtained in the presence of 10–11 M luminal ANG II as we increased [CO2]BL from 0% CO2 (solution 5) to 20% CO2 (solution 8) at a fixed [HCO3–]BL of 22 mM and a fixed pHBL of 7.4. The diamond at 5% CO2 represents the same data that we already presented in Fig. 3A (see bar labeled the 10–11 M). The circles summarize the control data in the absence of added ANG II. These control data are the same as those presented in Fig. 2A. The statistical analysis of these JHCO3 data was part of the same JHCO3 ANOVA discussed in conjunction with Fig. 2A. Compared with the control condition, 10–11 M luminal ANG II produced a statistically significant increase in JHCO3 at 0% (P = 0.0023) and 5% CO2 (P = 0.011). However, the difference was not statistically significant at a [CO2]BL of 20% (P = 0.055). Viewed differently, low-dose luminal ANG II produces a modest upward shift of the relationship between JHCO3 and [CO2]BL and low [CO2]BL values but eliminated the stimulation by 20% CO2.
The statistical analysis of the JV data was part of the same JV ANOVA discussed in conjunction with Fig. 2B. Compared with the control situation with no added hormone (circles in Fig. 4B), low-dose luminal ANG II (diamonds) produced a statistically significant increase in JV (diamonds) at 0% CO2 (P = 0.0020) but did not have a significant effect at 5 (P = 0.14) or 20% CO2 (P = 0.99).
Effect of high-dose ANG II. The squares in Fig. 4A summarize JHCO3 data obtained in the presence of 10–9 M luminal ANG II as we increased [CO2]BL from 0% CO2 to 20% CO2 at a fixed [HCO3–]BL of 22 mM and a fixed pHBL of 7.4. The statistical analysis of these JHCO3 data was part of the same JHCO3 ANOVA discussed in conjunction with Fig. 2A and the diamonds in Fig. 4A. Compared with the control condition, high-dose luminal ANG II had no effect on JHCO3 at 0% CO2 (P = 1.00) or 5% (P = 0.34), but produced a statistically significant decrease at 20% CO2 (P < 0.0001). Viewed differently, high-dose basolateral ANG II flattened the relationship between JHCO3 and [CO2]BL.
The statistical analysis of these JV data was part of the same JV ANOVA discussed in conjunction with Fig. 2B and the diamonds in Fig. 4B. Compared with the control situation with no added hormone (circles in Fig. 4B), high-dose luminal ANG II (diamonds) produced a statistically significant increase in JV at 0% (P = 0.0075) and 5% CO2 (P = 0.014). The hormone did not have a statistically effect at 20% CO2 (P = 0.55).
DISCUSSION
Effects of basolateral or luminal ANG II with equilibrated CO2/HCO3– solutions in the bath. It is well established that ANG II is a potent modulator of volume and HCO3– reabsorption by the PT. ANG II, regardless of whether it is added to the basolateral or to the luminal solution, tends at low doses to increase JV and JHCO3, but tends at high doses to decrease both parameters. Low-dose ANG II (pM range) appears to act via AT1 receptors (22, 24). Indeed, an AT1 antagonist blocks the effect of low-dose luminal ANG II on JV and JHCO3 in rabbit PTs (2). Moreover, low-dose luminal ANG II fails to increase JHCO3 in AT1A-deficient mice (42). The AT1 receptors apparently stimulate phospholipase C (PLC), which releases diacylglycerols that in turn activate protein kinase C. The role of inositol-1,4,5-trisphosphate (IP3), which ought to be released together with diacylglycerols, is not clear. Although it has also been suggested that ANG II enhances HCO3– reabsorption by lowering intracellular levels of cAMP (27), the consensus seems to be that ANG II produces its physiological effects without altering [cAMP]i (13, 18).
High-dose ANG II (nM-μM range), like low-dose ANG II, appears to act via AT1A receptors: high-dose luminal ANG II fails to reduce JHCO3 in AT1A-deficient mice (42). Cumulative evidence suggests that, at least in part, high-dose ANG II acts via PLA2 to release arachidonic acid (AA). In the epoxygenase pathway, a cytochrome P-450 enzyme then converts this AA to a metabolite such as 5,6-EET (3, 21, 24).
Our data obtained with the equilibrated CO2/HCO3– solution, [CO2]BL = 5%, [HCO3–]BL = 22 mM, and pHBL 7.4, generally confirm earlier observations that both basolateral and luminal ANG II have biphasic effects on JHCO3. We believe that ours is the first study to examine the effects of low- or high-dose basolateral ANG II on JHCO3 in a rabbit PT.
Mutual interdependence of the effects of basolateral ANG II and basolateral CO2 on JHCO3. As shown in Fig. 2A, 10–11 M basolateral ANG II tends to stimulate HCO3– reabsorption at low values of [CO2]BL but actually produces a small inhibition at the highest [CO2]BL. The upper curve in Fig. 5A is a replot of these data and confirms the general trend that, as [CO2]BL rises, the fractional stimulation produced by low-dose ANG II tends to fall, eventually turning into a small inhibition. Returning to Fig. 2A, we recall that 10–9 M basolateral ANG II inhibits HCO3– reabsorption at all values of [CO2]BL. The lower curve in Fig. 5A, a replot of these data, suggests that, as [CO2]BL rises, the fractional inhibition produced by high-dose ANG II is at first stable and then tends to increase at the highest [CO2]BL. In other words, under conditions in which the JHCO3 response to the CO2-sensing mechanism is greatest, low-dose ANG II produces the least stimulation and high-dose ANG II produces the greatest inhibition.
Another way to analyze our data is to ask how changes in [CO2]BL affect the tubule's response to ANG II. The black curve in Fig. 6A is a replot of the data at [CO2]BL = 5% in Fig. 2A and confirms the well-known biphasic effect of basolateral ANG II under "control" basolateral acid-base conditions. The red curve in Fig. 6A is a replot of the data at [CO2]BL = 0% in Fig. 2A and shows that, even with minimal stimulation of the basolateral CO2-sensing mechanism, basolateral ANG II also has a biphasic effect on JHCO3. Finally, the green curve in Fig. 6A is a replot of the data at [CO2]BL = 20% in Fig. 2A and shows that, with maximal stimulation of the basolateral CO2-sensing mechanism, basolateral ANG II no longer has a biphasic effect on JHCO3: both low-dose and high-dose ANG II now inhibit HCO3– reabsorption, with the effect being substantially more pronounced at 10–9 M.
Mutual interdependence of the effects of luminal ANG II and basolateral CO2 on JHCO3. We found that luminal ANG II tends to produce effects similar to those of basolateral ANG II, although the details are different. In Fig. 4A we saw that 10–11 M luminal ANG II stimulates HCO3– reabsorption at 0 and 5% CO2. The upper curve in Fig. 5B, a replot of these data, confirms that, as [CO2]BL rises, low-dose ANG II produces a smaller fractional stimulation of HCO3– reabsorption. Focusing again on Fig. 4A, we see that 10–9 M luminal ANG II has no effect on JHCO3 at the two lowest [CO2]BL, but reduces JHCO3 at 20% [CO2]BL. Indeed, the lower curve in Fig. 5B confirms that the fractional inhibition produced by high-dose ANG II progressively increases as [CO2]BL increases. Thus, as was the case for basolateral ANG II (see Fig. 5A), low-dose luminal ANG II produces its smallest stimulation of HCO3– reabsorption, and high-dose ANG II produces its greatest inhibition, when the JHCO3 response to the CO2-sensing mechanism is maximal.
In Fig. 6B, the black curve is a replot of the data at [CO2]BL = 5% in Fig. 4A. These results verify the biphasic effect of luminal ANG II under "control" basolateral acid-base conditions. The red curve in Fig. 6B, a replot of the data at [CO2]BL = 0% in Fig. 4A, shows that, with minimal stimulation of the basolateral CO2-sensing mechanism, luminal ANG II has a blunted biphasic effect on JHCO3. That is, low-dose ANG II increases JHCO3 but high-dose ANG II has no effect. This result contrasts to that with basolateral ANG II (see red curve in Fig. 6A), which produces a full biphasic effect (i.e., inhibition at high-dose ANG II) at [CO2]BL = 0%. The green curve in Fig. 6B, a replot of the data at [CO2]BL = 20% in Fig. 4A, shows that, with maximal stimulation of the basolateral CO2-sensing mechanism, luminal ANG II still has a biphasic effect on JHCO3. This result contrasts to that with basolateral ANG II (see green curve in Fig. 6A), which loses its biphasic effect at [CO2]BL = 20%.
In conclusion, the mutual effects of 1) basolateral or luminal ANG II on the basolateral CO2 dependence of JHCO3 and 2) basolateral [CO2] on the basolateral or luminal ANG II dependence of JHCO3 suggest to us that the signal-transduction pathways for basolateral CO2 intersect or perhaps even merge with the signal-transduction pathways for 1) low-dose basolateral ANG II, 2) high-dose basolateral ANG II, 3) low-dose luminal ANG II, and 4) high-dose luminal ANG II.
Our results raise two additional issues concerning low- vs. high-dose ANG II. Obviously, the distinction between a "low" stimulatory and a "high" inhibitory dose of ANG II is somewhat arbitrary and may differ according to the species studied and experimental preparation employed. In addition, our data demonstrate that the distinction between low and high also depends on whether one is examining JHCO3 or JV. For example, at a [CO2]BL of 20%, 10–9 M luminal ANG II lowered JHCO3 (Fig. 3A) but had no significant effect on JV (Fig. 3B). Thus increasing levels of [ANG II]L may produce decreases in JHCO3 earlier than they produce decreases in JV. Finally, our data indicate that the distinction between low and high depends on [CO2]BL. Thus, at a [CO2]BL of 20%, 10–11 M basolateral ANG II actually inhibited HCO3– reabsorption (green curve in Fig. 6A).
GRANTS
This work was funded by National Institutes of Health Program Project Grant PO1-DK-17433. P. Bouyer was supported by a National Kidney Foundation fellowship.
ACKNOWLEDGMENTS
We thank D. Wong for computer support.
FOOTNOTES
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. Section 1734 solely to indicate this fact.
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ABSTRACT
Previous authors showed that, at low doses, both basolateral and luminal ANG II increase the proximal tubule's HCO3– reabsorption rate (JHCO3). Using out-of-equilibrium CO2/HCO3– solutions, we demonstrated that basolateral CO2 increases JHCO3. Here, we examine interactions between ANG II and CO2 in isolated, perfused rabbit S2 segments. We first used equilibrated 5% CO2/22 mM HCO3–/pH 7.40 in bath and lumen. At 10–11 M, basolateral (BL) ANG II increased JHCO3 by 41%, and luminal ANG II increased JHCO3 by 35%. At 10–9 M, basolateral ANG II decreased JHCO3 by 43%, whereas luminal ANG II was without effect. Second, we varied [CO2]BL from 0 to 20% at fixed [HCO3–]BL and pHBL. Fractional stimulation produced by BL 10–11 M ANG II falls when [CO2]BL exceeds 5%. Fractional inhibition produced by BL 10–9 M ANG II tends to rise when [CO2]BL exceeds 5%. Regarding luminal ANG II, fractional stimulation produced by 10–11 M ANG II fell monotonically as [CO2]BL rose from 0 to 20%. Fractional inhibition produced by 10–9 M ANG II rose monotonically with increasing [CO2]BL. Viewed differently, ANG II at 10–11 M tended to reduce stimulation by CO2, and at 10–9 M, produced an even greater reduction. In conclusion, the mutual effects of 1) ANG II on the JHCO3 response to basolateral CO2 and 2) basolateral CO2 on the JHCO3 responses to ANG II suggest that the signal-transduction pathways for ANG II and basolateral CO2 intersect or merge.
kidney; out-of-equilibrium solutions; acid-base; volume reabsorption
ONE OF THE MAJOR tasks of the kidneys is to participate, along with the lungs, in the regulation of the acid-base balance of the extracellular fluid. For example, it has long been appreciated that acute respiratory acidosis (i.e., a rise in PCO2 that causes a fall in pH) rapidly stimulates renal H+ secretion (8, 16). The proximal tubule (PT) plays a key role in this acid secretion. In addition to reabsorbing a near-isosmotic fluid that represents about two-thirds of the fluid filtered by the glomerulus, the PT reabsorbs 80% of the filtered HCO3– as follows. The PT cell actively secretes H+ into the tubule lumen (1, 6, 35) and uses this H+ to titrate filtered HCO3– in the lumen to CO2 and H2O, catalyzed by apical carbonic anhydrase IV (9, 36, 37). The newly formed CO2 and H2O then diffuse into the PT cells, where carbonic anhydrase II (36, 37) catalyzes the regeneration of H+ and HCO3–. The cell extrudes the H+ across the apical membrane via Na-H exchangers (4, 5, 30) and H+ pumps (20), while exporting the HCO3– across the basolateral membrane, mainly via the electrogenic Na/HCO3 cotransporter (7, 15, 32, 33).
Using out-of-equilibrium (OOE) CO2/HCO3– solutions to alter [CO2], [HCO3–], or [H+] one at a time (40, 41, 43), our laboratory demonstrated that, at least in regard to acute acid-base disturbances, H+ secretion by the PT responds not to changes in pH, but only to changes in basolateral [CO2] and [HCO3–] (43). Thus, in the case of acute respiratory acidosis, the PT cell senses the increase in blood [CO2] per se, which is a powerful stimulus for HCO3– reabsorption.
Perhaps the most powerful hormonal stimulus for HCO3– reabsorption is ANG II. The first report of the effects of ANG II on PT transport was in 1968 by Burg and Orloff (12) on isolated, perfused rabbit PTs. They noted that adding 2 x 10–6 M ANG II to the basolateral solution had no detectable effect on the rate of fluid absorption (JV). In a 1974 rat micropuncture study, Steven (38) reported that 2 x 10–5 M basolateral ANG II lowered JV, whereas 1 x 10–7 M had no effect. Harris and Young (23) later showed that basolateral ANG II has a biphasic effect on Na+ reabsorption in the rat PT, stimulating at low doses (10–12-10–10 M) and inhibiting at higher doses (3 x 10–7-3 x 10–6 M). Shuster and colleagues (34) in 1984 demonstrated a similar biphasic effect of basolateral ANG II on JV in isolated, perfused rabbit PTs, ruling out a role of sympathetic innervation on the JV response. In 1991, working in isolated, perfused rat proximal straight tubules (PSTs), Garvin (19) found that 10–10 M basolateral ANG II increases both JHCO3 and JV. At about the same time, Chatsudthipong and Chan (14) found that high levels of basolateral ANG II reduce JHCO3 in rats and that these effects are blocked by saralasin, an antagonist of ANG II receptors.
As far as the effects of luminal ANG II are concerned, in a 1988 micropuncture study, Liu and Cogan (26) showed that low-dose luminal ANG II (10–12-10–11 M) increases HCO3– reabsorption (JHCO3) even in a denervated rat kidney. The 1990 study on the rat PT by Wang and Chan (39) extended the earlier observations by showing that increasing levels of luminal ANG II have biphasic effects on JHCO3 as well as JV. Consistent with these last two papers, Morduchowicz et al. (29), working in brush-border membrane vesicles, showed that ANG II stimulates Na-H exchange. Li et al. (25) in 1994 and Du et al. (17) in 2003 extended the biphasic effects of ANG II on JV to the lumen of the isolated, perfused rabbit PT. In 1997, working in isolated perfused rabbit proximal convoluted tubule (PCTs), Baum et al. (2) found that, in the presence but not in the absence of a luminal ACE inhibitor, low-dose luminal ANG II increased both JV and JHCO3.
The pattern that emerges from the above work is that, whether applied to the luminal or basolateral surface of the PT, low-dose ANG II generally increases JV and JHCO3, whereas high-dose ANG II generally has the opposite effect. In fact, until our observation that basolateral CO2 is also a powerful stimulus for HCO3– reabsorption (43), low-dose ANG II was the single most powerful known stimulus. The purposes of the present study were to determine whether 1) the effects of basolateral CO2 and low-dose ANG II, added to either the bath (i.e., basolateral solution) or lumen (i.e., luminal solution), are additive and 2) high-dose ANG II antagonizes the stimulatory effects of CO2.
Our approach was to use OOE solutions to vary basolateral [CO2] from 0 to 20% while keeping basolateral [HCO3–] and pH fixed near their physiological values in isolated, perfused rabbit S2 PTs. We found that low-dose (i.e., 10–11 M) ANG II, whether added to the bath or lumen, maximally increased JHCO3 at low basolateral (BL) [CO2], but that the effects tended to wane at high values of [CO2]BL. High-dose ANG II (i.e., 10–9 M) added to the bath produced a uniform decrease in JHCO3, regardless of [CO2]BL. When added to the lumen, high-dose ANG II had no effect when [CO2]BL was 0%, but increasingly larger inhibitor effects at high values of [CO2]BL.
METHODS
Biological preparation. All experiments were carried out in "pathogen-free" female rabbits (New Zealand White, Elite, Covance, Denver, PA) according to procedures that were approved by the Yale Animal Care and Use Committee. We perfused the PTs using methods that were similar to those originally described by Burg et al. (10) and later modified by Baum et al. (2) and also by our laboratory (31, 41). Briefly, a rabbit weighing 1.4–2.0 kg was euthanized by a single overdose of 3 ml (20 mg) of intravenous pentobarbital sodium. An incision of the abdominal wall exposed the left kidney, which we rapidly removed and then cut into coronal slices that we kept in cold (4°C) modified Hanks' solution (solution 1 in Table 1). Microdissection of the slice was carried out by hand in the same solution under a dissection microscope, using fine forceps, to yield individual midcortical S2 segments 1.5–1.7 mm in length, as detailed in Ref. 41. We cannulated the perfusion end of the tubule using concentric holding, perfusion and exchange pipettes. We cannulated the collection end of the tubule using a holding pipette and collected samples using a calibrated collection pipette (volume 55 nl), as summarized in Fig. 1B of Ref. 41. The mean length of perfused tubules in our JHCO3/JV experiments, as measured with an eyepiece micrometer, was 1.22 ± 0.01 mm (n = 99 tubules), representing the distal end of the PCT. The mean luminal collection rate was 12.3 ± 0.1 nl/min (n = 198 collection periods). We perfused the basolateral side of the tubule (i.e., "bath") at 7 ml/min, with a solution at 37°C.
Experimental protocol and solutions. The experimental protocol was similar to that of a previous study (43) except for the addition of ANG II to the luminal or basolateral solutions. Table 1 lists the compositions of the solutions, all of which were identical to those used in the aforementioned study (43). In brief, we dissected PTs in Hanks' solution (solution 1) at 4°C. The luminal perfusate always was solution 2, which contained dialyzed 3H-methoxyinulin (MW 7146, catalogue no. NET-086L, PerkinElmer Life Sciences, Boston, MA) as the volume marker. Solution 3, which contained 2% albumin, flowed through the bath during a 20- to 30-min warm-up period at 37°C. Following the warm-up period, all experiments had two periods for the collection of luminal fluid. During the first of these, the bath contained solution 4, with or without ANG II. During the second collection period, the bath contained ANG II dissolved in solutions 4, 5, 6, 7, or 8. In experiments in which we perfused the lumen with ANG II, the hormone was present in the lumen throughout the experiment; however, in these experiments, ANG II was always absent from the bath during both collection periods. We generated OOE CO2/HCO3– solutions (solutions 5-8) by rapidly mixing streams of two dissimilar solutions (40) and delivering the newly mixed solution to the tubule within 200 ms. All solutions had osmolalities of 300 ± 2 mosmol/kgH2O.
Measurement of JHCO3 and JV. In each of the two collection periods, we allowed the tubule to stabilize in the appropriate bath solution for 5–8 min, removed and discarded the fluid that had accumulated in the holding pipette at the collection end of the tubule, and then began a series of three timed and calibrated collections. The first two were subsequently analyzed for [3H]methoxyinulin for use in the calculation of JV, and the third was analyzed for total CO2 for use in the calculation of JHCO3. Our measurement of JHCO3 (pmol·min–1·mm–1 tubule length) and JV (nl·min–1·mm–1) was similar to that used by McKinney and Burg (28) and identical to our previous approach (41, 43). We determined total CO2 in aliquots of the perfusate and collected fluid using a WPI "NanoFlo" device (World Precision Instruments, Sarasota, FL) together with Diagnostic Kit 132-A (Sigma, St. Louis, MO). In this paper, the values that we report for JHCO3 (or JV) in first collection period are unnormalized mean values. The values that we report for JHCO3 (or JV) in the second collection period are normalized, mean values computed as described previously (43). Briefly, in each experiment, we divided the JHCO3 (or JV) value obtained during the second collection period by the comparable values obtained during the first collection period; the result was a pair of second/first collection-period ratios. We then multiplied 1) the second/first ratio for JHCO3 (or JV) in a particular experiment by 2) the unnormalized, mean, JHCO3 (or JV) value that we obtained during the first collection periods in a series of experiments following the identical protocol.
Data analysis. For comparisons at 0, 5, and 20% basolateral CO2, we performed one-way ANOVA and Dunnett's multiple comparison for ANOVA for five means using KaleidaGraph (Version 4, Synergy Software, Reading, PA). In this analysis, which we applied separately for JHCO3 and JV at each level of [CO2]BL, we compared the control condition (no hormone) with basolateral 10–11 M ANG II, basolateral 10–9 M ANG II, luminal 10–11 M ANG II, and luminal 10–9 M ANG II. For comparisons at 2.5 and 10% basolateral CO2, we performed two-tailed, unpaired t-tests using the Analysis Toolpack of Microsoft Excel. Results are given as means ± SE, with the number of tubules (n) from which it was calculated.
RESULTS
Effects of basolateral ANG II on JHCO3 and JV with an equilibrated CO2/HCO3– solution in the bath. In our first set of studies, we exposed the basolateral surface of S2 PTs to an equilibrated CO2/HCO3– solution, [CO2]BL = 5%, [HCO3–]BL = 22 mM, and pHBL = 7.4 (solution 4), throughout the two collection periods of the experiment. During the first collection period, no hormone was present (Fig. 1A, open bar). During the second collection period, we added to the bath either 10–11 M ANG II (gray bar) or 10–9 M ANG II (filled bar). Thus we were able to compare the effects of ANG II on JHCO3 and JV in the same tubule. Because in our analysis of basolateral ANG II (Fig. 1A) we used the same control data as in our analysis of luminal ANG II (see Fig. 3A), we employed one-way ANOVA for five groups: 1) the identical control data for Figs. 1A and 3A; 2 and 3) the basolateral low- or high-dose ANG II data for Fig. 1A; and 4 and 5) the luminal low- or high-dose ANG II data for Fig. 3A. The overall P value was <0.0001. Dunnett's multiple comparison shows that, compared with the control condition in which no added ANG II was present in the bath, the presence of a "low dose" of 10–11 M ANG II increased JHCO3 significantly from 56 ± 3 to 79 ± 5 pmol·min–1·mm–1 (P < 0.0001), as shown by the first two bars in Fig. 1A. In contrast, adding a "high dose" of 10–9 M ANG II decreased JHCO3 significantly from 56 ± 3 to 33 ± 4 pmol·min–1·mm–1 (P < 0.0001).
Our control mean JHCO3 value of 56 pmol·min–1·mm–1 is similar to the values of 82 pmol·min–1·mm–1 reported by Burg and Green (11) and 62 pmol·min–1·mm–1 reported by Baum et al. (2), both from the isolated, perfused rabbit PCT. The 41% increase in JHCO3 that 10–11 M ANG II produced in our experiments is somewhat higher than the 29% increase that 10–10 M ANG II produced in the experiments in isolated, perfused rat PSTs by Garvin (19).
Figure 1B summarizes the JV data, which we analyzed using the same ANOVA approach summarized in the previous paragraph. The overall P value was 0.00013. The effect of 10–11 M basolateral ANG II on JV was stimulatory, as it was for JHCO3. The low dose of the peptide increased the mean JV from 0.46 ± 0.03 to 0.62 ± 0.05 nl·min–1·mm–1 (P = 0.0087). On the other hand, adding 10–9 M basolateral ANG II did not have a statistically significant effect on JV, which changed from 0.46 ± 0.03 under control conditions to 0.41 ± 0.05 nl·min–1·mm–1 in the presence of the high dose of the peptide (P = 0.8033).
Our control JV value of 0.46 nl·min–1·mm–1 is about two-thirds as great as the value reported by Schuster et al. (34), who worked with a combination of S1 and S2 segments from midcortical and juxtamedullary nephrons. On the other hand, our observed 36% increase of JV with 10–11 M ANG II is about twice as great as that observed by the earlier investigators. Our JV data confirm the earlier observations of others, made in a combination of rat (14, 23, 38) and rabbit (34) PTs, that increasing levels of basolateral ANG II have a biphasic effect on JV. In addition, we observed that increasing levels of basolateral ANG II have a biphasic effect on JHCO3. Our JHCO3 data are consistent with those made by Garvin (19) in rat PSTs for low-dose ANG II, and with those made in rats by Chatsudthipong and Chan (14) for high-dose ANG II.
Effects of basolateral ANG II on the basolateral CO2 dependence of JHCO3 and JV. A previous study from our laboratory demonstrated that increasing [CO2]BL from 0 to 20%, using OOE technology to fix [HCO3–]BL at 22 mM and fix pHBL at 7.4, substantially stimulated bicarbonate reabsorption by the rabbit S2 PT (43). In the present study, we examined the effects of low- or high-dose basolateral ANG II on the basolateral CO2 dependence of JHCO3. In this series of experiments, the bath contained 10–11 or 10–9 M ANG II plus an equilibrated 5% CO2/22 mM HCO3–/pH 7.40 solution during the first collection period, and an OOE solution containing the same level of ANG II and the same 22 mM HCO3–/pH 7.40 but a variable level of CO2 during the second collection period.
Effect of low-dose ANG II. The diamonds in Fig. 2A summarize JHCO3 data obtained in the presence of 10–11 M basolateral ANG II as we increased [CO2]BL from 0% CO2 (solution 5) to 20% CO2 (solution 8) at a fixed [HCO3–]BL of 22 mM and a fixed pHBL of 7.4. The circles summarize comparable control data in the absence of added ANG II. These control data are from an earlier study (43), augmented by 12 additional experiments at 5% CO2 from Fig. 1A. In the previous section, we discussed the use of one-way ANOVA for five groups to analyze the data at a [CO2]BL of 5%. We similarly used one-way ANOVA for five groups (Figs. 2A and 4A) to analyze the data at [CO2]BL values of 0 and 20%. The overall P values were <0.0001 for both the 0 and 20% data. In addition, we used a t-test to analyze the data at [CO2]BL values of 2.5 and 10% in Fig. 2A. Compared with the control condition, 10–11 M basolateral ANG II produced a statistically significant increase in JHCO3 at 0 (P = 0.041) and 5% CO2 (P < 0.0001). The difference was not statistically significant at either 2.5 (P = 0.092) or 10% CO2 (P = 0.51). Basolateral 10–11 M ANG II produced a small but statistically significant decrease in JHCO3 at 20% CO2 (P = 0.032). Viewed differently, low-dose basolateral ANG II steepens relationship between JHCO3 and [CO2]BL at low levels of [CO2]BL (i.e., 0–5%), but eliminated the stimulation by CO2 at higher [CO2]BL levels.
We analyzed the JV data the same way we analyzed the JHCO3 data. For the ANOVA, the overall P values were 0.00088 for 0% CO2 and 0.063 for 20% CO2. Compared with the control situation with no added hormone (circles in Fig. 2B), low-dose basolateral ANG II (diamonds) tended to increase JV, although the effect was statistically significant only at a [CO2]BL value of 5%.
Effect of high-dose ANG II. The squares in Fig. 2A summarize JHCO3 data obtained in the presence of 10–9 M basolateral ANG II as we increased [CO2]BL from 0% CO2 to 20% CO2 at a fixed [HCO3–]BL of 22 mM and a fixed pHBL of 7.4. The statistical analysis of these JHCO3 data was part of the same JHCO3 ANOVA discussed two paragraphs above. Compared with the control condition, high-dose basolateral ANG II produced a statistically significant decrease in JHCO3 at all three levels of [CO2]BL: 0% (P = 0.0049), 5% (P < 0.0001), and 20% (P < 0.0001). Viewed differently, high-dose basolateral ANG II flattens the relationship between JHCO3 and [CO2]BL at low levels of [CO2]BL (i.e., 0–5%) and eliminates CO2 sensitivity at higher [CO2]BL levels.
The statistical analysis of the JV data was part of the same JV ANOVA discussed two paragraphs above. Compared with the control situation with no added hormone (circles in Fig. 2B), high-dose basolateral ANG II (squares) had no significant effect on JV (Fig. 2B).
Effects of luminal ANG II on JHCO3 and JV with equilibrated CO2/HCO3– solutions in the bath. In our next set of studies, all of the data came from the first collection period of experiments. The open bar in Fig. 3A repeats the control JHCO3 data from Fig. 1A. The gray bar in Fig. 3A represents the effect on JHCO3 of perfusing the lumen with 10–11 M ANG II, and the filled bar represents the effect on JHCO3 of perfusing the lumen with 10–9 M ANG II. The statistical analysis of these JHCO3 data was part of the same JHCO3 ANOVA discussed in conjunction with Fig. 1A. Luminal 10–11 M ANG II significantly increased JHCO3 from 56 ± 3 to 76 ± 7 pmol·min–1·mm–1 (P = 0.011). In contrast, adding a "high dose" of 10–9 M ANG II caused JHCO3 to change by a statistically insignificant amount, from 56 ± 3 to 46 ± 4 pmol·min–1·mm–1 (P = 0.34).
The stimulation by 10–11 M ANG II that we observed confirms the observation by others in rat PTs by Wang and Chan (39). These same authors found that 10–8 M luminal ANG II reduced JHCO3 in rat, whereas we observed no significant effect at 10–9 M. In rabbit PCTs, Baum et al. (2) found no effect of luminal ANG II at concentrations from 10–11 to 2 x 10–8 M. However, in the presence of luminal enalaprilat, these authors found that 10–10 M luminal ANG II did indeed increase JHCO3. Two technical differences between our study and that of Baum et al. is that they perfused the bath at 0.5 ml/min (vs. 7.0 ml/min) and added 6 g/dl albumin to the bath throughout the experiment (vs. 2 g/dl only during the warm-up period).
The statistical analysis of the JV data in Fig. 3B was part of the same JV ANOVA discussed in conjunction with Fig. 1B. Luminal 10–11 M ANG II changed the mean JV by a statistically insignificant amount from 0.46 ± 0.03 to 0.60 ± 0.06 nl·min–1·mm–1 (P = 0.14). Others had observed a stimulation by low-dose ANG II on rabbit PTs (17, 25). We found that adding 10–9 M luminal ANG II significantly increased JV from 0.46 ± 0.03 to 0.67 ± 0.06 nl·min–1·mm–1 (P = 0.014), which is in a direction opposite that seen by others in the rabbit (17, 25) or rat (39).
Effects of luminal ANG II on the basolateral CO2 dependence of JHCO3 and JV. In this series of studies, the lumen contained 10–11 or 10–9 M ANG II throughout the experiment. During the first collection period, the bath contained equilibrated 5% CO2/22 mM HCO3–/pH 7.40, while during the second collection period the bath contained an OOE solution with the same 22 mM HCO3–/pH 7.40 but a variable level of CO2.
Effect of low-dose ANG II. The diamonds in Fig. 4A summarize JHCO3 data obtained in the presence of 10–11 M luminal ANG II as we increased [CO2]BL from 0% CO2 (solution 5) to 20% CO2 (solution 8) at a fixed [HCO3–]BL of 22 mM and a fixed pHBL of 7.4. The diamond at 5% CO2 represents the same data that we already presented in Fig. 3A (see bar labeled the 10–11 M). The circles summarize the control data in the absence of added ANG II. These control data are the same as those presented in Fig. 2A. The statistical analysis of these JHCO3 data was part of the same JHCO3 ANOVA discussed in conjunction with Fig. 2A. Compared with the control condition, 10–11 M luminal ANG II produced a statistically significant increase in JHCO3 at 0% (P = 0.0023) and 5% CO2 (P = 0.011). However, the difference was not statistically significant at a [CO2]BL of 20% (P = 0.055). Viewed differently, low-dose luminal ANG II produces a modest upward shift of the relationship between JHCO3 and [CO2]BL and low [CO2]BL values but eliminated the stimulation by 20% CO2.
The statistical analysis of the JV data was part of the same JV ANOVA discussed in conjunction with Fig. 2B. Compared with the control situation with no added hormone (circles in Fig. 4B), low-dose luminal ANG II (diamonds) produced a statistically significant increase in JV (diamonds) at 0% CO2 (P = 0.0020) but did not have a significant effect at 5 (P = 0.14) or 20% CO2 (P = 0.99).
Effect of high-dose ANG II. The squares in Fig. 4A summarize JHCO3 data obtained in the presence of 10–9 M luminal ANG II as we increased [CO2]BL from 0% CO2 to 20% CO2 at a fixed [HCO3–]BL of 22 mM and a fixed pHBL of 7.4. The statistical analysis of these JHCO3 data was part of the same JHCO3 ANOVA discussed in conjunction with Fig. 2A and the diamonds in Fig. 4A. Compared with the control condition, high-dose luminal ANG II had no effect on JHCO3 at 0% CO2 (P = 1.00) or 5% (P = 0.34), but produced a statistically significant decrease at 20% CO2 (P < 0.0001). Viewed differently, high-dose basolateral ANG II flattened the relationship between JHCO3 and [CO2]BL.
The statistical analysis of these JV data was part of the same JV ANOVA discussed in conjunction with Fig. 2B and the diamonds in Fig. 4B. Compared with the control situation with no added hormone (circles in Fig. 4B), high-dose luminal ANG II (diamonds) produced a statistically significant increase in JV at 0% (P = 0.0075) and 5% CO2 (P = 0.014). The hormone did not have a statistically effect at 20% CO2 (P = 0.55).
DISCUSSION
Effects of basolateral or luminal ANG II with equilibrated CO2/HCO3– solutions in the bath. It is well established that ANG II is a potent modulator of volume and HCO3– reabsorption by the PT. ANG II, regardless of whether it is added to the basolateral or to the luminal solution, tends at low doses to increase JV and JHCO3, but tends at high doses to decrease both parameters. Low-dose ANG II (pM range) appears to act via AT1 receptors (22, 24). Indeed, an AT1 antagonist blocks the effect of low-dose luminal ANG II on JV and JHCO3 in rabbit PTs (2). Moreover, low-dose luminal ANG II fails to increase JHCO3 in AT1A-deficient mice (42). The AT1 receptors apparently stimulate phospholipase C (PLC), which releases diacylglycerols that in turn activate protein kinase C. The role of inositol-1,4,5-trisphosphate (IP3), which ought to be released together with diacylglycerols, is not clear. Although it has also been suggested that ANG II enhances HCO3– reabsorption by lowering intracellular levels of cAMP (27), the consensus seems to be that ANG II produces its physiological effects without altering [cAMP]i (13, 18).
High-dose ANG II (nM-μM range), like low-dose ANG II, appears to act via AT1A receptors: high-dose luminal ANG II fails to reduce JHCO3 in AT1A-deficient mice (42). Cumulative evidence suggests that, at least in part, high-dose ANG II acts via PLA2 to release arachidonic acid (AA). In the epoxygenase pathway, a cytochrome P-450 enzyme then converts this AA to a metabolite such as 5,6-EET (3, 21, 24).
Our data obtained with the equilibrated CO2/HCO3– solution, [CO2]BL = 5%, [HCO3–]BL = 22 mM, and pHBL 7.4, generally confirm earlier observations that both basolateral and luminal ANG II have biphasic effects on JHCO3. We believe that ours is the first study to examine the effects of low- or high-dose basolateral ANG II on JHCO3 in a rabbit PT.
Mutual interdependence of the effects of basolateral ANG II and basolateral CO2 on JHCO3. As shown in Fig. 2A, 10–11 M basolateral ANG II tends to stimulate HCO3– reabsorption at low values of [CO2]BL but actually produces a small inhibition at the highest [CO2]BL. The upper curve in Fig. 5A is a replot of these data and confirms the general trend that, as [CO2]BL rises, the fractional stimulation produced by low-dose ANG II tends to fall, eventually turning into a small inhibition. Returning to Fig. 2A, we recall that 10–9 M basolateral ANG II inhibits HCO3– reabsorption at all values of [CO2]BL. The lower curve in Fig. 5A, a replot of these data, suggests that, as [CO2]BL rises, the fractional inhibition produced by high-dose ANG II is at first stable and then tends to increase at the highest [CO2]BL. In other words, under conditions in which the JHCO3 response to the CO2-sensing mechanism is greatest, low-dose ANG II produces the least stimulation and high-dose ANG II produces the greatest inhibition.
Another way to analyze our data is to ask how changes in [CO2]BL affect the tubule's response to ANG II. The black curve in Fig. 6A is a replot of the data at [CO2]BL = 5% in Fig. 2A and confirms the well-known biphasic effect of basolateral ANG II under "control" basolateral acid-base conditions. The red curve in Fig. 6A is a replot of the data at [CO2]BL = 0% in Fig. 2A and shows that, even with minimal stimulation of the basolateral CO2-sensing mechanism, basolateral ANG II also has a biphasic effect on JHCO3. Finally, the green curve in Fig. 6A is a replot of the data at [CO2]BL = 20% in Fig. 2A and shows that, with maximal stimulation of the basolateral CO2-sensing mechanism, basolateral ANG II no longer has a biphasic effect on JHCO3: both low-dose and high-dose ANG II now inhibit HCO3– reabsorption, with the effect being substantially more pronounced at 10–9 M.
Mutual interdependence of the effects of luminal ANG II and basolateral CO2 on JHCO3. We found that luminal ANG II tends to produce effects similar to those of basolateral ANG II, although the details are different. In Fig. 4A we saw that 10–11 M luminal ANG II stimulates HCO3– reabsorption at 0 and 5% CO2. The upper curve in Fig. 5B, a replot of these data, confirms that, as [CO2]BL rises, low-dose ANG II produces a smaller fractional stimulation of HCO3– reabsorption. Focusing again on Fig. 4A, we see that 10–9 M luminal ANG II has no effect on JHCO3 at the two lowest [CO2]BL, but reduces JHCO3 at 20% [CO2]BL. Indeed, the lower curve in Fig. 5B confirms that the fractional inhibition produced by high-dose ANG II progressively increases as [CO2]BL increases. Thus, as was the case for basolateral ANG II (see Fig. 5A), low-dose luminal ANG II produces its smallest stimulation of HCO3– reabsorption, and high-dose ANG II produces its greatest inhibition, when the JHCO3 response to the CO2-sensing mechanism is maximal.
In Fig. 6B, the black curve is a replot of the data at [CO2]BL = 5% in Fig. 4A. These results verify the biphasic effect of luminal ANG II under "control" basolateral acid-base conditions. The red curve in Fig. 6B, a replot of the data at [CO2]BL = 0% in Fig. 4A, shows that, with minimal stimulation of the basolateral CO2-sensing mechanism, luminal ANG II has a blunted biphasic effect on JHCO3. That is, low-dose ANG II increases JHCO3 but high-dose ANG II has no effect. This result contrasts to that with basolateral ANG II (see red curve in Fig. 6A), which produces a full biphasic effect (i.e., inhibition at high-dose ANG II) at [CO2]BL = 0%. The green curve in Fig. 6B, a replot of the data at [CO2]BL = 20% in Fig. 4A, shows that, with maximal stimulation of the basolateral CO2-sensing mechanism, luminal ANG II still has a biphasic effect on JHCO3. This result contrasts to that with basolateral ANG II (see green curve in Fig. 6A), which loses its biphasic effect at [CO2]BL = 20%.
In conclusion, the mutual effects of 1) basolateral or luminal ANG II on the basolateral CO2 dependence of JHCO3 and 2) basolateral [CO2] on the basolateral or luminal ANG II dependence of JHCO3 suggest to us that the signal-transduction pathways for basolateral CO2 intersect or perhaps even merge with the signal-transduction pathways for 1) low-dose basolateral ANG II, 2) high-dose basolateral ANG II, 3) low-dose luminal ANG II, and 4) high-dose luminal ANG II.
Our results raise two additional issues concerning low- vs. high-dose ANG II. Obviously, the distinction between a "low" stimulatory and a "high" inhibitory dose of ANG II is somewhat arbitrary and may differ according to the species studied and experimental preparation employed. In addition, our data demonstrate that the distinction between low and high also depends on whether one is examining JHCO3 or JV. For example, at a [CO2]BL of 20%, 10–9 M luminal ANG II lowered JHCO3 (Fig. 3A) but had no significant effect on JV (Fig. 3B). Thus increasing levels of [ANG II]L may produce decreases in JHCO3 earlier than they produce decreases in JV. Finally, our data indicate that the distinction between low and high depends on [CO2]BL. Thus, at a [CO2]BL of 20%, 10–11 M basolateral ANG II actually inhibited HCO3– reabsorption (green curve in Fig. 6A).
GRANTS
This work was funded by National Institutes of Health Program Project Grant PO1-DK-17433. P. Bouyer was supported by a National Kidney Foundation fellowship.
ACKNOWLEDGMENTS
We thank D. Wong for computer support.
FOOTNOTES
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. Section 1734 solely to indicate this fact.
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