2Division of Renal Diseases and Hypertension, University of Texas, Medical School at Houston, Houston, Texas 77030; and 1Renal Division, Emory University School of Medicine, Atlanta, Georgia 30322
Submitted 12 November 2002 ; accepted in final form 30 June 2003
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ABSTRACT |
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ammonium; acidification; angiotensin 2; aldosterone; metabolic acidosis; metabolic alkalosis
In the OMCD, secretion of net H+ equivalents is thought to occur across the type A intercalated cell. Immunolocalization studies in rat and rabbit have shown that the H+-ATPase and Cl-/ exchange (AE1) are present in type A intercalated cells at opposite plasma membrane domains (1). With changes in acid-base balance, the distribution of the H+-ATPase is changed within the cell. After NH4Cl ingestion, a model of metabolic acidosis, expression of H+-ATPase in the apical plasma membrane increases in tandem with increased AE1 immunoreactivity in the basolateral plasma membrane (5, 25). Increased expression of these transporters along the plasma membrane of the OMCD and cortical collecting duct (CCD) increases net acid secretion and thereby contributes to restoration of acid-base balance.
However, the effect of NH4Cl ingestion on net acid secretion in OMCD tubules perfused in vitro has been the subject of controversy. Studies in rat (3) and rabbit (19, 33) have concluded that total CO2 flux (JtCO2) is either unchanged or undergoes an adaptive increase after ingestion of NH4Cl.
NH4Cl administration is associated not only with metabolic acidosis but also with a number of hormonal changes, such as increased plasma renin and aldosterone levels (26). Whether changes in the renin-angiotensin system, which occur after intake of NH4Cl, change JtCO2 is unknown. Moreover, the effect of ANG II on net acid secretion in the medullary collecting duct is unknown. The purpose of the present study was to determine in rat OMCD 1) the effect of increased intake of H+ equivalents on net acid secretion and 2) to determine if aldosterone and ANG II, which increase after NH4Cl ingestion, alter H+ secretion.
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METHODS |
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Collecting ducts from the inner stripe of the outer medulla (OMCD; see Ref. 9) were dissected from pathogen-free male Sprague-Dawley rats weighing 65120 g (Rm. 205G; Harlan, Indianapolis, IN). Animals were housed in microisolator cages and fed a 0.07% Na+ and 0.8% K+ diet (Zeigler Brothers, Garners, PA; see Ref. 37).
In Vivo Treatment Protocols
Series 1. Rats received 50 mM NH4Cl, NaCl, or NaHCO3 in their drinking water for 7 days before death. Controls drank tap water only. With the use of this protocol, intake of NH4Cl and NaHCO3 varies between 8.08 and 8.39 µeq·g-1·day-1 (24).
Series 2. Rats received 160 mM NH4Cl, NaCl, or NaHCO3 in their drinking water for 7 days before death. Controls drank tap water only. In each of these groups, we observed that rats ingest the following (in µeq · g-1 · day-1): NH4Cl, 36.1 ± 7.6 (n = 5); NaCl, 36.9 ± 2.0 (n = 5); and NaHCO3, 35.4 ± 3.2 (n = 5).
Series 3. Rats received 5 mg deoxycorticosterone pivalate (DOCP; CIBA-Geigy Animal Health, Greensboro, NC) by intramuscular injection 59 days before death. Both untreated rats and rats receiving DOCP drank tap water ad libitum.
Series 4. Rats received 5 mg DOCP (CIBA-Geigy Animal Health) by intramuscular injection 59 days before death. Both untreated rats and rats receiving DOCP drank tap water containing 50 mM NaHCO3 ad libitum for 7 days before death.
Series 5. Rats were anesthetized with 4% isofluorane and O2 at 1 l/min. Osmotic minipumps were implanted subcutaneously (Alzet, Palo Alto, CA) to deliver 0.1 or 10 ng/min ANG II (in 160 mM NaCl) by continuous infusion for 59 days before death. Controls received vehicle only (160 mM NaCl in water) through the minipump over the same time interval. Both controls and rats receiving ANG drank 50 mM NaHCO3 for 7 days before death.
All animals were injected with furosemide (5 mg/100 g body wt ip) 45 min before death by decapitation to attenuate changes in the extracellular osmolality of the tubule (37). Coronal slices were cut from the kidneys and placed in a dish containing the chilled experimental solution (11°C). The dissection solution was identical to that used in the lumen and bath in perfusion experiments. To isolate OMCD's from the inner stripe (9), a cut was made between the inner and outer stripe of the outer medulla. The OMCD inner stripe and inner medulla were transferred to a second dish for dissection. Tubules were mounted on concentric glass pipettes and perfused in vitro at 37°C.
Experiments were performed with symmetric solutions in the bath and perfusate. Solution composition was as follows (in mM): 121 NaCl, 5 KCl, 4 NH4Cl, 25 NaHCO3,1Na2HPO4, 2 CaCl2, 1.2 MgSO4, and 5.5 glucose (solution 1); 144 NaCl, 5 KCl, 6 NH4Cl, 1 Na2HPO4, 2 CaCl2, 1.2 MgSO4, 5.5 glucose, and 1 HEPES, pH 7.4 (solution 2); and 125 NaCl, 5 KCl, 25 NaHCO3, 1 Na2HPO4, 2 CaCl2, 1.2 MgSO4, and 5.5 glucose (solution 3). Osmolality was measured in all solutions (37). To maintain the desired CO2 concentration in - and/or CO2-buffered solutions, the perfusate was passed through jacketed concentric tubing through which 95% air-5% CO2 was blown in a countercurrent direction around the perfusate line (37, 41). To maintain pH in
-containing solutions, the bath fluid was bubbled constantly with 95% air-5% CO2. In HEPES-buffered solutions, bath fluid was bubbled with 100% O2. When comparing flux in the presence and absence of inhibitors, experimental groups were alternated. Moreover, each experimental protocol was performed in more than one shipment of rats. For in vitro experiments, rat ANG II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe; Sigma, St. Louis, MO; see Ref. 22) was prepared as a 10-4 M stock in water. Candesartan (CV11974) was prepared as a 10-2 M stock solution. This candesartan stock solution also contained 156 mM NaCl and 12.5 mM Na2CO3.
Because of time-dependent changes in transepithelial potential difference and total ammonia flux (JtAMM; see Ref. 39), measurements were performed under a single experimental condition in each tubule studied. All measurements were begun 30 min, and terminated 75 min, after warming the tubule. The mean rate of fluxes measured over this time period was reported. For flux measurements, perfused fluid was collected continuously as a series of equal-volume samples.
Measurement of and JtAMM
Tubule fluid samples were collected under oil in calibrated constriction pipettes. Flow rate was determined as described previously (41) and varied between 1 and 4 nl · mm-1 · min-1. However, when comparing flux between treatment groups, no difference in flow rate was detected between these groups. Total CO2 concentration was measured in the collected fluid and perfusate using a continuous-flow fluorimeter (29, 37). This method can detect (total CO2) concentration differences of <1 mM with an 8-nl pipette (29, 37).
absorption, JtCO2, was calculated from the luminal flow rate and the total CO2 (tCO2) concentration difference measured in the perfusate and collected fluid (37). A sample of the perfusate solution from the perfusate reservoir was placed under water-equilibrated mineral oil. Samples of the perfusate and collected fluid were alternated. Net fluid transport in the OMCD was taken to be zero in the absence of an imposed osmolality gradient (40).
Total ammonia concentration (tAMM; see Refs. 37 and 41) was measured in collected perfusate samples, as described previously, using a continuous-flow fluorimeter. This total ammonia assay can distinguish differences in total ammonia concentration of 0.1 mM using an 11-nl pipette. JtAMM determinations were made as described above for tCO2.
Measurement of Systolic Blood Pressure and Arterial pH
Systolic blood pressure was measured in conscious rats by tail cuff using an MOD 59 Pulse Amplifier (Innovators in Instrumentation, Woodland Hills, CA) or a BP-2000 (Visitech Systems, Apex, NC). To measure arterial pH, rats were anesthetized as described above, and arterial blood was drawn from the abdominal aorta in a heparinized syringe. Arterial pH, PO2, and PCO2 were measured with an IL 1620 (Instrumentation Laboratories, Lexington, MA) or an AVL OPTl 1 Blood Gas Analyzer (AVL Medical Instruments, Saint-Ouen L'GAumone, France).
Statistical Analysis
When measuring tAMM or tCO2, one to four replicate measurements were made for each tubule. The flux reported for each tubule represents the mean of all replicate measurements made for that tubule. When only one measurement was made, that value was reported for the tubule. Thus the "n" reported equals the number of tubules studied. Only one tubule was studied from a given rat. When comparing two groups, statistical significance was determined using an unpaired two-tailed Student's t-test. Statistical significance was achieved with a P < 0.05. Comparisons between four groups were made using ANOVA with a Bonferoni posttest. Statistical significance was achieved with a P < 0.017. Data are displayed as means ± SE.
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RESULTS |
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To determine the effect of changes in acid-base homeostasis on net acid secretion, rats drank 50 mM NH4Cl, NaHCO3, or NaCl for 7 days (series 1). Other animals drank tap water only over the same time period. Between these treatment groups, urinary ammonium excretion varies 10-fold (24), although arterial pH is similar (Table 1). Thus arterial pH is maintained within a narrow range despite huge variations in intake of H+ or OH- equivalents (Table 1) through the regulated excretion of ammonium (38).
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The OMCD is critical in the regulation of ammonium excretion (36). Thus changes in net acid secretion, which occur after changes in intake of H+ or OH- equivalents, were studied further in rat OMCD. JtCO2 or JtAMM was measured in OMCD tubules from rats in each treatment group given in series 1. Tubules were perfused in vitro in symmetric - and/or CO2-buffered solutions (solution 1). Under these conditions, absorption of
and secretion of ammonium in the OMCD were substantial, as reported previously (10). As shown, JtCO2 was similar in OMCD tubules from rats drinking NaHCO3, NaCl, or water alone (Fig. 1). However, JtCO2 was reduced by 55% (P < 0.017) in animals drinking NH4Cl relative to those drinking NaHCO3. Because this was a surprising result, we asked if ammonium secretion (JtAMM) in OMCD is increased in rats ingesting NH4Cl relative to JtAMM measured in rats ingesting less H+ equivalents. Figures 1 and 2 show that JtAMM did not increase in tubules from animals ingesting NH4Cl relative to tubules from animals drinking water alone or animals ingesting equimolar concentrations of NaCl or NaHCO3. NH4Cl ingestion failed to increase JtAMM even when the intake of H+ or OH- equivalents in these groups was increased threefold. Moreover, ingestion of NH4Cl did not increase JtAMM in OMCD tubules when measured either in the presence (solution 1) or the absence1 (solution 2)of
/CO2. Therefore, with NH4Cl ingestion, net acid secretion in rat OMCD is reduced paradoxically.
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Effect of In Vivo Aldosterone and ANG II on JtCO2
Because plasma aldosterone and ANG II greatly augment acid secretion in other segments, and because these hormones should increase after ingestion of NH4Cl (13, 26), we tested the effect of aldosterone and ANG II in vivo on JtCO2 in OMCD tubules when perfused in vitro. Experiments were performed in rats, which drank water containing 50 mM NaHCO3 to suppress endogenous renin production and to increase JtCO2 in the OMCD (series 4 and 5; see Ref. 8). ANG II (0.1 ng/min) and DOCP (5 mg) produced a similar increase in systolic BP (Table 2) but produced different effects on acid-base balance. Although DOCP administration produced a metabolic alkalosis (Table 2), no change in arterial pH was observed at either a high (10 ng/min) or a low (0.1 ng/min) dose of ANG II (Table 2). Thus the effect of these hormones on net acid secretion in the OMCD was studied further. DOCP administration (series 4, Fig. 3) did not affect JtCO2.2 However, ANG II at 0.1 ng/min decreased JtCO2 by 34% (P < 0.05, Fig. 3). A decrease in JtCO2 was not detected at 10 ng/min ANG II. However, these data show that ANG II did not increase JtCO2 at a higher dose (10 ng/min, Table 2). We conclude that low doses of ANG II in vivo reduce JtCO2 in vitro without changing arterial pH.
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Effect of ANG II In Vitro on JtCO2
To determine if ANG has a direct effect on acid secretion in rat OMCD, JtCO2 was measured when these hormones were applied to the bath solution. OMCD tubules from rats drinking NaHCO3 (series 1) were perfused and bathed in the presence of solution 1. ANG II (10-8 M) in the bath reduced JtCO2 by 35% (P < 0.05, Fig. 4). This ANG II-mediated effect was abolished with the AT1 receptor antagonist candesartan (10-6 M) present in the bath solution (Fig. 4). No effect of AT II was detected at a concentration of 10-10 M [10.5 ± 1.7 pmol · mm-1 · min-1 in controls, n = 5, vs. 8.24 ± 1.5 pmol · mm-1 · min-1, n = 5, with 10-10 M ANG II present in the bath solution, P = not significant (NS)]. Thus ANG II inhibits JtCO2 in vitro through AT1 receptors. Because aldosterone increases JtCO2 in OMCD in vitro (31), the effect of 10-9 M aldosterone, when applied to the bath, on JtCO2 was measured. In the absence of the hormone, JtCO2 was 10.0 ± 0.5 pmol · mm-1 · min-1 (n = 4) and 7.42 ± 1.5 pmol · mm-1 · min-1 (n = 6) in the presence of 10-9 M aldosterone in the bath (P = NS).
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DISCUSSION |
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Despite a dramatic upregulation of H+ secretion in the proximal and distal tubule by ANG II, we observed that infusion of ANG II did not produce a metabolic alkalosis. Thus compensatory regulation of acid secretion must occur in other segments of the nephron after an ANG II infusion. Although ANG II upregulates H+ secretion in the proximal and distal tubule, ANG II reduces H+ secretion in other segments when perfused in vitro. In the rat medullary thick ascending limb, 10-8 M ANG II in the bath solution reduces absorption by 40% (11). In the outer portion of the rabbit CCD, 10-7 M ANG II increases
secretion (43). Therefore, in the medullary thick ascending limb and the CCD, ANG II reduces net acid secretion.
The present study shows that ANG II reduces absorption in the OMCD when applied in vitro. Because the ANG II-induced reduction in JtCO2 was blocked by candesartan, this effect is mediated through AT1 receptors and also demonstrates that AT1 receptors are present along the basolateral membrane of rat OMCD. This observation is compatible with previous reports that AT1 message is expressed in rat OMCD (21).
Which H+ transporters are regulated by ANG II in rat OMCD remains to be determined. It is likely, however, that ANG II in vitro reduces H+- and ATPasemediated H+ secretion in the rat OMCD. In rat CCD (32) and inner medullary collecting duct (34), ANG II reduces H+-ATPase activity. The concentration dependence of ANG II for changes in JtCO2, determined in the present study, is similar to that reported previously for ANG II and the H+-ATPase in rat CCD by Tojo, Tisher, and Madsen (32). In both the Tojo study (32) and in the present study, a decrease in H+ transport was observed at an ANG II concentration of 10-8 M, but not at 10-10 M. Moreover, in both studies, the effect of ANG II on H+ transport was abolished with AT1 receptor blockade.
The present study also shows that ANG II reduces JtCO2 in OMCD when applied both in vitro and in vivo. The effect of ANG II in vivo on JtCO2 may occur through a direct effect of ANG II on AT1 receptors, as it does in vitro. However, we cannot exclude the possibility that the effect of ANG II on JtCO2 observed when applied in vivo is indirect, such as through changes in sympathic nerve activity (17). Alternatively, ANG II may alter net acid secretion in the OMCD through changes in distal Na+ delivery.3 Whether AT1 receptors localize to the apical plasma membrane and whether activation of these receptors by ANG II modulates JtCO2 remains to be determined.
In vivo, ANG II produces a variety of effects such as increasing production of mineralocorticoid by the adrenal gland. However, it is unlikely that 0.1 nl/min ANG II reduces JtCO2 through increased circulating mineralocorticoid levels. First, the reduction in JtCO2 observed with ANG II administration was not reproduced with DOCP. Results of the present study are consistent with previous studies that have shown that DOCP administration either produces no effect or increases H+ secretion (14, 31). Thus administration of an aldosterone analog has an effect opposite to that we observed after ANG II infusion. Second, aldosterone synthesis is stimulated at relatively high levels of circulating ANG II (44). Thus our inability to detect a reduction in JtCO2 at higher doses of ANG II (10 ng/min) may be because of the resulting synthesis of aldosterone, which modulates the effect of ANG II on JtCO2. Although a decrease in JtCO2 at 10 ng/min ANG II was not detected, these data demonstrate that ANG II does not produce a biphasic response at higher doses.
Because ANG II and aldosterone levels generally increase in tandem, it is possible that the effect of aldosterone opposes the effect of ANG II in vitro at levels observed in the renal interstitium in vivo. Previous studies in rabbit OMCD by Stone and collaborators (31) have shown that 10-8 to 10-6 M aldosterone, when applied to the bath solution, stimulates JtCO2 in vitro through an Na+-independent mechanism. However, circulating levels of aldosterone are generally in the range of 10-11 to 10-9 M (12, 13, 20), concentrations much lower than those employed in the study by Stone et al. (31). The complete dose-response effect of aldosterone on JtCO2 in the rat OMCD over the physiological range of circulating aldosterone concentration is the subject of future studies. However, at 10-9 M aldosterone, we did not observe an increase in JtCO2 in rat OMCD.
Concentrations of ANG II (10-8 M) that reduce net acid secretion in the rat OMCD in vitro are two to three orders of magnitude above ANG II levels reported previously in systemic plasma of the rat (8.9 x 10-11 M; see Ref. 27). However, ANG II levels in star vessel plasma, which reflects interstitial ANG II concentration in rat cortex, are 10-8 to 10-7 M (11, 27). Because tissue ANG II content is higher in medulla than in cortex, interstitial ANG II levels in rat outer medulla are probably higher than concentrations in the cortex (23). This hypothesis cannot be tested directly, however, because the interstitium of the rat outer medulla is not accessible to micropuncture. Nevertheless, concentrations of ANG II (10-8 M) that reduce JtCO2 in rat OMCD in vitro fall within the physiological range of ANG II levels observed in the kidney in vivo.
The physiological significance of the segment-specific response of H+ secretion to ANG II remains to be determined. It is well established that ANG II infusion increases Na+ absorption by the kidney, which helps maintain intravascular volume in many physiological/pathophysiological conditions (11). In the proximal tubule, Na+ serves as a counter-ion for H+ section. Thus an ANG II infusion augments H+ secretion and Na+ absorption. In more distal nephron segments, ANG II also augments Na+ absorption (7). One can hypothesize therefore that, by reducing H+ secretion in the distal nephron, while stimulating H+ secretion in the proximal nephron, ANG II markedly stimulates Na+ retention, while producing little change in systemic pH (11). Because the OMCD is thought to mediate 4050% of net acid secreted along the collecting duct (36), this ANG II-mediated effect in this segment is likely to be important physiologically.
The present study demonstrates with NH4Cl ingestion that JtCO2 is reduced when measured in vitro. This observation is consistent with previous reports in rats by Atkins and Burg (3) and in rabbits by Lombard et al. (19). In both studies, JtCO2 in the OMCD was lower in animals ingesting NH4Cl than in animals drinking either water alone or drinking water containing NaHCO3 (Table 3), although these differences did not achieve statistical significance. However, in rabbit OMCD, Tsuruoka and Schwartz (33) reported an increase in JtCO2 after NH4Cl ingestion (Table 3). The reason for these different results is not clear.
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The decrease in acid secretion observed in the OMCD from rats ingesting NH4Cl was a surprising result, since maintaining acid-base balance in this treatment model requires increased excretion of H+ equivalents by the kidney. After NH4Cl ingestion, an intrinsic, stable adaptation may occur in the OMCD that reduces H+ secretion, while restoring balance of another cation, e.g., K+. K+ absorption in the OMCD occurs through a passive mechanism, driven by its chemical gradient (K+ recycling; see Ref. 15). K+ absorption must occur in exchange for another cation or in parallel with absorption of an anion. In OMCD, K+/H+ exchange has been reported (2, 45). Thus increased H+ secretion might augment K+ absorption. Conversely, decreased H+ secretion might reduce K+ absorption and attenuate hyperkalemia, which occurs during metabolic acidosis. We cannot exclude the possibility that the effect of NH4Cl ingestion on H+ secretion in the OMCD differs when measured in vitro vs. when measured in vivo. Thus the physiological significance of the decrease in net acid secretion in rat OMCD observed with NH4Cl ingestion remains to be determined.
In conclusion, ANG II applied both in vivo and in vitro reduces JtCO2 in rat OMCD. The increase in circulating ANG II expected after NH4Cl ingestion might contribute to the decrease in JtCO2 observed in vitro in rat OMCD.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
1 Because the hydration of CO2 provides the primary H+ source in rat OMCD (39), we have observed that JtAMM falls by 75% upon the removal of
/CO2.
2 The effect of DOCP on JtCO2 in rat OMCD was confirmed by varying the experimental conditions. OMCD tubules from rats receiving DOCP were compared with untreated rats (series 3). Both groups drank water only. Tubules were perfused and bathed in solution 3. JtCO2 was 6.3 ± 1.4 pmol · mm-1 · min-1 (n = 5) in controls and 7.3 ± 0.6 pmol · mm-1 · min-1 (n = 6) in rats receiving DOCP (P = NS). Thus administration of DOCP in vivo did not affect JtCO2. In rat OMCD perfused in vitro in symmetric - and/or CO2-buffered solutions, JtCO2 falls by
20% upon NH4Cl removal from the bath and perfusate (39). NH4Cl increases JtCO2 because NH3 buffers secreted H+ (39).
3 ANG II should reduce distal Na+ delivery, which may decrease net H+ secretion in the OMCD. Conversely, administration of loop diuretics should increase distal delivery of Na+, which may stimulate net H+ secretion in this segment. The effect of furosemide on net acid secretion in the rat OMCD is beyond the scope of the current study. Moreover, if furosemide administration in vivo alters net H+ secretion in vitro, this effect was carried over into all of the treatment groups studied and therefore should not affect the conclusions of the present study.
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REFERENCES |
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