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Role of endogenously secreted angiotensin II in the CO₂-induced stimulation of HCO₃ reabsorption by renal proximal tubules

Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut

Submitted 10 April 2007 ; accepted in final form 28 September 2007

	ABSTRACT

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Previous studies demonstrated that the proximal tubule (PT) responds to isolated increases in basolateral ([CO₂]_BL) or "bath" CO₂ concentration by increasing the HCO₃^– reabsorption rate (J). Blockade of the rabbit apical AT₁ receptor or knockout of the mouse AT_1A receptor eliminates these effects, demonstrating a requirement for luminal ANG II that the PT itself synthesizes. In the present study, we examined the effects of the ACE inhibitor lisinopril on J in isolated perfused rabbit PTs (S2 segment), using out-of-equilibrium solutions to make isolated changes in [CO₂]_BL at a fixed baseline HCO₃^– concentration of 22 mM and fixed baseline pH of 7.4. Adding 60 or 240 nM lisinopril (in vitro K_i: 0.5 or 1.2 nM) to the lumen had no effect. These results are not consistent with the hypothesis that the PT secretes either angiotensinogen or ANG I. However, adding 60 nM basolateral lisinopril significantly decreased J at a [CO₂]_BL of 20%. Moreover, 240 nM basolateral lisinopril decreased baseline (i.e., at 5% CO₂) J by one-half and completely eliminated the response to altering [CO₂]_BL from 0 to 20%, but left intact the stimulatory effect of 10^–11 M basolateral ANG II. At extremely high concentrations (i.e., 100 µM), luminal lisinopril replicated the effects of 240 nM basolateral lisinopril. Our data are consistent with the hypothesis that lisinopril readily crosses the basolateral (but not apical) membrane to block ACE in a vesicular compartment. We conclude that the isolated PT predominantly secretes preformed ANG II, rather than angiotensinogen or ANG I.

kidney; renin-angiotensin system; lisinopril; angiotensin-converting enzyme; acid-base

The classic endocrine renin-angiotensin system (RAS) plays an important role in regulating cardiovascular function (7, 13, 21). Moreover, the elements of the classic RAS are well understood: the secretion of angiotensinogen by the liver, the secretion of renin by juxtaglomerular cells in the kidney, the conversion by renin of angiotensinogen to ANG I, the presence of angiotensin-converting enzyme (ACE) on endothelial cells, the conversion by ACE of ANG I to ANG II, and the actions of ANG II on the vasculature, kidney, and adrenal cortex.

Investigators also have identified several examples of "local" or "tissue" RAS (3, 20), operating more or less autonomously from the classic endocrine RAS and using tissue-specific mechanisms to regulate local ANG II levels, in a wide range of tissues, such as the central nervous system, heart, vasculature, and reproductive tract. Indeed, the first local system to be elucidated was the intrarenal RAS (4, 15). The PT cell has all the components for its own RAS (4), including angiotensinogen, renin, ANG I, and brush-border ACE (8, 28), to generate local ANG II (5, 19). Our earlier work (34) indicates that the RAS in the PT plays at least a critical permissive role in the J response to alterations in [CO₂]_BL.

In the present study, we tested the hypotheses that 1) the PT secretes not ANG II but either angiotensinogen or ANG I, and therefore 2) blocking apical ACE (i.e., preventing ANG I ANG II) should be equivalent to blocking apical AT₁ receptors. We examined the effect of the ACE inhibitor lisinopril, added to either the lumen or bath (i.e., basolateral solution), on the CO₂-induced stimulation of HCO₃^– reabsorption in renal proximal tubules. Our approach was to perfuse isolated S2 segments from the rabbit and to use out-of-equilibrium (OOE) solutions to vary [CO₂]_BL from 0 to 20% while keeping [HCO₃^–]_BL and pH_BL fixed near their physiological values. Surprisingly, we found that submicromolar levels of lisinopril had no effect on J when we added the drug to the lumen but reduced baseline J(i.e., the value when [CO₂]_BL = 5%) and eliminated the [CO₂]_BL sensitivity of J when we added the drug to the bath. These results indicate that the proximal tubule secretes mainly preformed ANG II, rather than an ANG II precursor, into the tubule lumen and suggest that the lisinopril added to the bath acts by blocking the intracellular conversion of ANG I to ANG II.

	METHODS

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Biological preparation. "Pathogen-free" female rabbits (New Zealand White, Elite; Charles River Laboratories, Wilmington, MA) were used in the present study. The procedures were approved by the Yale Animal Care and Use Committee. In brief, our approach for obtaining and perfusing rabbit proximal tubules was similar to that originally described by Burg et al. (2) and later modified by Baum et al. (1) and also by our laboratory (18, 31). Rabbits (1.4–2.0 kg) were euthanized with pentobarbital sodium (40 mg), given as a single intravenous overdose. After surgically exposing and removing the left kidney, we obtained coronal sections, which we incubated in Hanks' solution (solution 1 in Table 1). We then microdissected slices, isolating individual midcortical S2 PT segments, each 1.5–1.7 mm long (31). Using concentric holding, perfusion, and exchange pipettes, we cannulated the "perfusion end" of the tubule, randomly selecting (and recording the choice) either the more proximal or more distal end of the tubule. We cannulated the "collection end" of the tubule using a holding pipette. Finally, we collected samples using a calibrated collection pipette (volume 55 nl), as summarized in Fig. 1 B in Ref. 31.

Experimental protocol and solutions. Our protocol was virtually identical to the one that we used in previous studies (32–35) except that, in the present study, we added lisinopril (a gift from Merck, Rahway, NJ), a specific ACE inhibitor, to the luminal or basolateral solution.

Listed in Table 1 are the compositions of the solutions, which are identical to those used in our most recent study (34). The luminal perfusate was always solution 2, containing dialyzed [³H]methoxyinulin (PerkinElmer Life Sciences, Boston, MA) as a volume marker. During a 20- to 30-min warm-up period, solution 3 at 37°C flowed through the bath. Afterward, during the actual experiment, solution 4 (equilibrated CO₂/HCO₃^–) flowed through the bath during the first of the two collection periods. During the second of the two collection periods, solution 5 or 6 flowed through the bath. These two OOE CO₂/HCO₃^– solutions had a [CO₂]_BL of 0 or 20%, respectively, but a "normal" [HCO₃^–]_BL (i.e., 22 mM) and pH_BL (i.e., 7.40). We rapidly mixed solutions 5A and 5B to yield solution 5, and we rapidly mixed solutions 6A and 6B to yield solution 6. The osmolality was 300 ± 2 mosmol/kgH₂O for all solutions.

Measurement of J_HCO3 and J_V. We measured J(pmol·min^–1·mm^–1 tubule length) and J_V (nl·min^–1·mm^–1) using an approach similar to that of McKinney and Burg (16). In aliquots of the perfusate and collected fluid, we determined total CO₂ using a WPI "NanoFlo" device (World Precision Instruments, Sarasota, FL) in combination with reagents assembled in house, as described in Ref. 33.

As described previously (35), the reported mean values of J(or J_V) for the first of two collection periods (i.e., in which the bath solution contained the "control" equilibrated 5% CO₂/HCO₃^–/pH 7.40) are simple averages (i.e., not normalized). The reported values for the second of two collection periods (i.e., in which bath solution contained the "experimental" OOE solution) were normalized as in our previous studies (32–35). First, we divided the J(or J_V) value for the second collection period by the J(or J_V) value for the first collection period in that tubule, obtaining a pair of ratios (one for J and one for J_V). Second, we multiplied the J(or J_V) ratio by the unnormalized meanJ(or J_V) value obtained during the first collection period in all experiments of the same protocol (i.e., with or without luminal lisinopril).

We made statistical comparisons between historical J or J_V control data (i.e., in the absence of any inhibitors) and corresponding new data presented in this study using a one-way ANOVA for three groups (i.e., all data for [CO₂]_BL values of 0, 5, and 20%), followed by Dunnett's multiple comparison (i.e., for more than two means), using KaleidaGraph (version 4; Synergy Software). Results are means ± SE, with the number of tubules (n) from which it was calculated.

	RESULTS

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Effects of 60 and 240 nM luminal lisinopril on the basolateral CO₂ dependence of J_HCO3 and J_V. In our first set of experiments, we tested our hypotheses that the PT secretes angiotensinogen and/or ANG I into the lumen and that ACE at the extracellular surface of the brush border ultimately converts ANG I to ANG II (8). According to the product data sheet provided by Merck, the in vitro IC₅₀ value for lisinopril is 1.2 nM. Deddish et al. (6) reported that the IC₅₀ value of lisinopril for human renal ACE is 0.47 nM. In our experiments, we perfused both the lumen and the bath with equilibrated 5% CO₂/22 mM HCO₃^– solutions at pH 7.40, adding 60 nM lisinopril to the lumen in some experiments.

The open bar in Fig. 1A summarizes our control (i.e., drug free) J data on 28 rabbit S2 PTs from the present study. The filled bar in Fig. 1A summarizes comparable J data, obtained on four tubules in the presence of 60 nM luminal lisinopril, which is 50- to 120-fold greater than the published in vitro IC₅₀ values. We stopped collecting data at 60 nM lisinopril when we observed no obvious inhibition in the presence of 5% CO₂. The effect of 60 nM luminal lisinopril is not statistically significant.

View larger version (11K): [in this window] [in a new window]   Fig. 1. Effect of 60 nM luminal lisinopril on the HCO3– reabsorption rate (J ; A) and fluid reabsorption rate (JV; B). During the single collection period, the lumen contained solution 2 (open bars) or solution 2 plus 60 nM lisinopril (filled bars), whereas the bath contained solution 4. Values are means ± SE, with nos. of tubules in parentheses. We performed 1-way ANOVA for all data in Figs. 1–5. Dunnett's multiple comparison indicates that 60 nM luminal lisinopril had no effect on either J or JV. In A, for a postulated difference of mean of 20 pmol·min–1·mm–1, the computed statistical power is 96%.

View larger version (11K): [in this window] [in a new window]   Fig. 2. Effects of 240 nM luminal lisinopril on the basolateral [CO2] dependence of J (A) and JV (B). The luminal solution was solution 2 (open circles) or solution 2 plus 240 nM lisinopril (filled circles) throughout the entire experiment. The basolateral solutions were solution 4 (5% CO2) during the first collection period and solution 5 (0% CO2) or solution 6 (20% CO2) during the second collection period. In a 1-way ANOVA for 3 groups (all data in Figs. 1–5), the overall Pvalue was 0.44 for a baseline CO2 concentration ([CO2]BL) of 0% and <0.0001 for [CO2]BL of 5 and 20%. Dunnett's multiple comparison indicates that none of the J or JV values represented by black symbols (present study) is significantly different from the corresponding historical controls (shaded symbols). See supplementary material for more details. All supplementary material is available in the online version of this article.

Figures 1B and 2B summarize the J_V data. The statistical analysis shows that none of the J_V values is significantly different from the historical control at the same [CO₂]_BL.

Effects of 60 and 240 nM basolateral lisinopril on the basolateral CO₂ dependence of J_HCO3 and J_V. Our first two sets of experiments indicate that, when added to the lumen, neither 60 nM (Fig. 1) nor 240 nM lisinopril (Fig. 2) has an effect on the basolateral CO₂ dependence of J and J_V. In our third set of experiments, we examined the effect of basolateral lisinopril (in the absence of luminal lisinopril). The control data (Fig. 3A, open circles) are the same as in Fig. 2A. When we saw that 60 nM basolateral lisinopril (Fig. 3A, filled triangle) reduced J, but not quite to the "pedestal" level that we normally observe at 0% CO₂, we stopped collecting data at this lisinopril concentration.

View larger version (12K): [in this window] [in a new window]   Fig. 3. Effects of 60 and 240 nM basolateral lisinopril on the basolateral [CO2] dependence of J (A) and JV (B). The luminal solution was solution 2 (without lisinopril) throughout the experiment for all tubules. The basolateral solution during the first collection period was always solution 4 (5% CO2) without lisinopril (i.e., control). During the second collection period, the bath contained solution 5 (0% CO2) ± 240 nM lisinopril, solution 4 (5% CO2) + 240 nM lisinopril, or solution 6 (20% CO2) ± 60 or 240 nM lisinopril. Values are means ± SE, with nos. of tubules in parentheses. In a 1-way ANOVA for 3 groups (all data in Figs. 1–5), the overall Pvalue was 0.44 for a [CO2]BL of 0% and <0.0001 for [CO2]BL of 5 and 20%. Dunnett's multiple comparison indicated that some of the J values, represented by black symbols (present study), were significantly different from the corresponding historical controls (shaded symbols; **P < 0.01). See supplementary material for more information.

Our limited experiments at 60 nM basolateral lisinopril provided an important clue that lisinopril might cross the basolateral membrane to inhibit the intracellular ACE-like activity. If this hypothesis is correct, then increasing the concentration of basolateral lisinopril ought to lead to higher intracellular levels of the drug, and thus to a greater inhibition of J. Therefore, in our fourth set of experiments, we followed the same protocol as above but increased the basolateral concentration of lisinopril to 240 nM. As summarized by the filled diamonds in Fig. 3A, 240 nM basolateral lisinopril appeared to have no effect on J at a [CO₂]_BL of 0% (solution 5), which remained at the pedestal level, but markedly reduced J at [CO₂]_BL values of 5% (solution 4) and 20% (solution 6) of the pedestal level.

Thus, 240 nM basolateral lisinopril reduces baseline J at 5% CO₂ and eliminates the J response to alterations in [CO₂]_BL. Furthermore, at a [CO₂]_BL of 20%, basolateral lisinopril produces a dose-dependent decrease in J(compare filled triangle vs. filled diamond in Fig. 3A). The most straightforward explanation for these data is that lisinopril enters the PT cell across the basolateral membrane and, by blocking intracellular ACE, prevents the secretion of the preformed ANG II that is required for the stimulation of HCO₃^– reabsorption by basolateral CO₂.

The filled diamonds in Fig. 3B summarize the J_V data obtained with 240 nM basolateral lisinopril and show that the drug had no significant effect on J_V at this concentration.

Effects of luminal 100 µM lisinopril on the basolateral CO₂ dependence of J_HCO3 and J_V. The preceding data show that whereas nanomolar levels of lisinopril, when added to the bath, substantially inhibit (filled triangle in Fig. 3A) or block (filled diamonds in Fig. 3A) the CO₂-dependent component of J, these same levels of lisinopril fail to reduce J when added to the lumen (Fig. 1A and Fig. 2A). The most straightforward explanation for these results is that ACE has already converted ANG I to ANG II before the enzyme reaches the brush border and that lisinopril does not readily cross the apical membrane of PT to inhibit intracellular ACE. If this hypothesis is true, then raising the luminal lisinopril concentration to levels that others have used with enalaprilat (22) might raise the apical lisinopril influx sufficiently to inhibit intracellular ACE and thereby reduce J.

To test the above hypothesis, we followed the protocol of Fig. 2 but raised the luminal lisinopril concentration to 100 µM. The filled squares in Fig. 4A summarize the effect of 100 µM luminal lisinopril on J and show that the response was virtually identical to that produced by 240 nM lisinopril when added to the bath. That is, at a [CO₂]_BL of 0%, J remained at the pedestal level, and at [CO₂]_BL levels of 5 and 20%, J fell to the pedestal level. The control data (open circles in Fig. 4A) are the same as in Figs. 2A and 3A. These data are consistent with the hypothesis that at extremely high luminal concentrations of lisinopril, the drug enters the PT cell at sufficiently high rates to block intracellular ACE-like activity, presumably doing so by directly entering the cell across the apical membrane or by crossing the tight junctions and then entering across the basolateral membrane.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Effects of 100 µM luminal lisinopril on the basolateral [CO2] dependence of J (A) and JV (B). The luminal solution was solution 2 (open circles) or solution 2 plus 100 µM lisinopril (filled squares) throughout the entire experiment. The basolateral solutions were solution 4 (5% CO2) during the first collection period, and solution 5 (0% CO2) or solution 6 (20% CO2) during the second collection period. Values are means ± SE, with nos. of tubules in parentheses. In a 1-way ANOVA for 3 groups (all data in Figs. 1–5), the overall P value was 0.44 for a [CO2]BL of 0% and <0.0001 for [CO2]BL of 5% and 20%. Dunnett's multiple comparison indicated that some of the J values represented by black symbols (present study) are significantly different from the corresponding historical controls (shaded symbols; **P < 0.01). See supplementary material for more information.

Effects of 10^–11 M ANG II in the presence of 240 nM basolateral lisinopril. A trivial explanation for the data in Fig. 3A is that 60 nM basolateral lisinopril, and even more so, 240 nM basolateral lisinopril, merely causes a nonspecific blockade of acid-base transport. If the inhibitory effect of lisinopril is truly limited to the blockade of ANG II production, leaving intact the ANG II receptors and the downstream signal-transduction processes and transporters, then we ought to be able to overcome the inhibitory effect of lisinopril by adding exogenous ANG II. Previous investigators (9, 10) as well as we (32) have shown that low-dose basolateral ANG II increases J in PTs. Therefore, with equilibrated 5% CO₂/22 mM HCO₃^– in both the lumen and bath, we examined the effect of adding 10^–11 M ANG II to the bath in the presence of 240 nM basolateral lisinopril.

As we have already shown in Fig. 3A, 240 nM basolateral lisinopril significantly reduces J(Fig. 5A, lightly shaded vs. open bar), whereas 10^–11 M ANG II in the presence of basolateral 240 nM lisinopril still raises J(darkly shaded bar) to virtually the same level as in previous experiments in which we added the ANG II in the absence of an ACE inhibitor (32). Thus, even though 240 nM basolateral lisinopril blocks the tubule's J sensitivity to basolateral CO₂, this drug leaves the tubule susceptible to stimulation by basolateral ANG II.

View larger version (13K): [in this window] [in a new window]   Fig. 5. Effects of 10–11 M ANG II in the presence of 240 nM basolateral lisinopril. A: J . B: JV. The luminal solution was solution 2. The basolateral solutions were solution 4 (5% CO2) during the first collection period and solution 4 (5% CO2) + 240 nM lisinopril ± 10–11 M ANG II during the second collection period. The data represented by the open bars are replotted from Figs. 1–4 ([CO2]BL = 5%), and the data represented by the light shaded bars are replotted from Fig. 3 (filled diamond at [CO2]BL = 5%). Values are means ± SE, with nos. of tubules in parentheses. In a 1-way ANOVA (all data for [CO2]BL = 5% in Figs. 1–5), the overall P value was <0.0001. Dunnett's multiple comparison indicates that the J values represented by light and dark shaded bars are significantly different from the controls represented by the open bars (**P < 0.01). See supplementary material for more information.

	DISCUSSION

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Intrarenal RAS. The renal PT cell contains all of the components necessary for a local RAS (4). For example, PT cells are positive for angiotensinogen mRNA (12), compartmentalize angiotensinogen protein in a membrane-bound compartment (11), and are positive for the protein at the brush border (23). Moreover, cultured PT cells secrete immunoreactive angiotensinogen into the apical fluid (23). Note, however, that the detected protein could include, or consist entirely of, remnant angiotensinogen (i.e., after cleavage of ANG I).

Renin mRNA is present in cultured rabbit PT cells, and renin activity is present in lysates from these cells (17). In PT cells, renin protein is in granular and vesicular compartments (26), where it colocalizes with angiotensinogen protein (11). In addition, cultured PT cells secrete reninlike activity into the culture medium (29).

The ANG I concentration in the PT fluid of anesthetized rats is higher than in the blood plasma (19), consistent with the hypothesis that PT cells secrete ANG I. Using an immunodetection assay, Hunt et al. (11) obtained evidence for the secretion of ANG I by renal cortical cells, although they could not rule out the secretion of ANG II, and demonstrated that the extracellular addition of a peptidic renin inhibitor had no effect on this secretion. These last results strongly support the hypothesis that PT cells convert angiotensinogen to ANG I within a vesicular compartment in the secretory pathway (Fig. 7).

View larger version (15K): [in this window] [in a new window]   Fig. 6. Effects of 240 nM basolateral and 100 µM luminal lisinopril on calculated parameters. A: J and concentrations of other solutes (JOther) in the absence of inhibitors. J data represent both the historical and present control data; JOther data were calculated from the corresponding J and JV values as (2 x J+ JOther)/JV = 300 mosmol/kgH2O (35). B: calculated concentrations of NaHCO3 ([NaHCO3] = J/JV) and other solutes ([Other] = JOther/JV) in the reabsorbate in the absence of inhibitors. C: J and JOther in the presence of 240 nM basolateral or 100 µM luminal lisinopril. J data are the same data plotted in Fig. 3A for 240 nM basolateral lisinopril or in Fig. 4A for 100 µM luminal lisinopril. D: calculated [NaHCO3] and [Other] in the reabsorbate in the presence of 240 nM basolateral or 100 µM luminal lisinopril. We made statistical comparisons among data represented by open circles (in A and B) and filled diamonds or filled squares (in C and D) by applying a 1-way ANOVA for each level of [CO2]BL. *P < 0.05; **P < 0.01.

View larger version (52K): [in this window] [in a new window]   Fig. 7. Model of the endogenous secretion of ANG II. We suggest that inside intracellular vesicles, the proximal tubule (PT) converts the majority of angiotensinogen (Agt) to ANG I and then to ANG II. In terms of angiotensin-related products, the fusion of these vesicles with the PT apical membrane would result in the secretion mainly of preformed ANG II and remnant angiotensinogen (rAgt). The PT would also secrete renin and deliver angiotensin-converting enzyme (ACE) to the brush border. Escaping vesicular peptidase activity may be small amounts of Agt and ANG I, which could then be cleaved by renin and ACE in the lumen of the PT or later nephron segments.

Not only do the PT and certain other nephron segments have all the components necessary for a local RAS, but the intrarenal RAS can play an important functional role in modulating both vascular parameters, such as renal plasma flow and glomerular filtration rate (for reviews, see Refs. 4, 14), and tubule parameters, including the reabsorption of Na⁺ and fluid (1, 22) as well as HCO₃^– (1, 34).

Effect of lisinopril on J_HCO3. Our previous study (34) demonstrated that either blocking the apical AT₁ receptor with candesartan in rabbits or knocking out the AT_1A receptor in mice eliminated the PT's J response to changes in [CO₂]_BL. These results confirm that the PT can somehow deliver ANG II to the PT lumen and that this luminal ANG II acts in an autocrine fashion. However, neither our earlier study nor those of others could distinguish among three possible options for luminal ANG II delivery: 1) The tubule could secrete angiotensinogen and renin into the lumen, while the vesicular membrane simultaneously delivers ACE to the brush border. The secreted renin could convert the angiotensinogen to ANG I, and the exocytosed ACE could convert the ANG I to ANG II. 2) The tubule could convert angiotensinogen to ANG I in a vesicular compartment and then secrete renin, remnant angiotensinogen, and preformed ANG I into the lumen, while the vesicular membrane simultaneously delivers ACE to the brush border. The exocytosed ACE would convert the ANG I to ANG II. 3) The tubule could convert angiotensinogen to ANG I and then convert ANG I to ANG II, all in a vesicular compartment. The PT cell then would secrete renin, remnant angiotensinogen, the NH₂-terminal dipeptide from ANG I, and preformed ANG II into the lumen, while the vesicular membrane would simultaneously deliver ACE to the brush border.

Of course, options 2 and 3 could be more complicated if the vesicular renin could not convert all of the angiotensinogen to ANG I or if the vesicular ACE could not convert all of the ANG I to ANG II. The physiological effect of any secreted angiotensinogen would obviously depend on the rate of luminal ANG II synthesis (which would depend on the local luminal concentration of angiotensinogen, luminal renin activity, and the apical ACE activity), the linear velocity of fluid along the tubule lumen (which would carry angiotensinogen and ANG I to more distal sites), the concentration of preexisting luminal ANG II (recall that ANG II has a biphasic effect on tubule transport), and the rate of ANG II degradation, all of which would likely vary with distance along the nephron. Similar logic applies to the physiological effect of any secreted ANG I. Thus it is possible that if the PT secretes angiotensinogen or ANG I, some of the secreted material might not have a physiological action (after conversion to ANG II) until it reaches the distal nephron.

Our initial hypotheses were option 1, followed by option 2. However, if most of the luminal ANG II arises from the conversion of ANG I to ANG II by brush-border ACE, then adding lisinopril to the lumen should have markedly lowered luminal [ANG II] and thus reduced baseline J and eliminated the sensitivity of J to changes in [CO₂]_BL. In our initial experiments, we applied 60 nM lisinopril to the lumen, a concentration that is 50- to 120-fold higher than the in vitro IC₅₀ value. However, in four initial experiments, this concentration, which should have blocked 98–99% of accessible ACE according to published values for the in vitro IC₅₀ of lisinopril, had no effect on J(Fig. 1A). Moreover, when we increased luminal lisinopril concentration to 240 nM, which should have blocked 99.5–99.8% of accessible ACE, the lisinopril still had no effect on either baseline J or sensitivity of J to changes in [CO₂]_BL from 0 to 20% at a fixed [HCO₃^–]_BL of 22 mM and a fixed pH_BL of 7.4 (Fig. 2A). Only when we raised luminal lisinopril concentration to 100 µM (Fig. 4A), which should have blocked 99.999% of accessible ACE, did we observe a substantial effect, consistent with earlier results obtained with the luminal application of enalaprilat (22). We consider it highly unlikely that blocking 99.999% vs. 99.5–99.8% of the brush-border conversion of ANG I to ANG II should have produced an appreciable difference in luminal [ANG II], and thus a noticeable difference in J.

These data are consistent with two hypotheses: 1) The conversion of most ANG I to ANG II does indeed take place at the brush border, but PT ACE is unusually insensitive to lisinopril. However, the IC₅₀ value of 0.47 nM obtained by Deddish et al. (6) argues that renal ACE is extremely sensitive to lisinopril. 2) PT cells convert most ANG I to ANG II in a vesicular compartment and secrete preformed ANG II into the lumen. According to this latter view: a) lisinopril added to the lumen blocks brush-border ACE only after the enzyme has already converted ANG I to ANG II, b) lisinopril fails to block vesicular ACE because the inhibitor cannot efficiently cross the apical membrane, and c) the inhibitor can efficiently cross the basolateral membrane.

If hypothesis 1 were true and PT ACE had an unusually low sensitivity to lisinopril, then we should not have observed substantial inhibition of J until the basolateral lisinopril concentration had reached extremely high levels. However, we found that applying as little as 60 nM lisinopril in the bath reduced J at a [CO₂]_BL of 20% from 89 to 57 pmol·min^–1·mm^–1(filled triangle in Fig. 3A), which represents an approximately two-thirds inhibition of the flux above the pedestal level of 32 pmol·min^–1·mm^–1. Raising basolateral lisinopril to 240 nM reduced J to the pedestal level at [CO₂]_BL levels from 0% to 20% (filled diamonds in Fig. 3A). Thus, consistent with the IC₅₀ value of Deddish et al. (6), our data indicate that PT ACE is reasonably sensitive to the drug and therefore that hypothesis 1 must be incorrect. We conclude that the PT secretes mainly intact ANG II (i.e., hypothesis 2).

To rule out the possibility that lisinopril nonspecifically blocks PT acid-base transport, we asked whether, in the presence of 240 nM basolateral lisinopril, 10^–11 M basolateral ANG II could still raise J. Indeed, Fig. 5A demonstrates that PT can still respond to basolateral ANG II even though it cannot respond to basolateral CO₂. Thus we propose that the J response to basolateral CO₂ requires the luminal secretion of preformed ANG II.

Effect of lisinopril on the flux of "other" solutes. In an earlier study (34), we found that the acute blockade of apical angiotensin AT receptors by saralasin, or of AT₁ receptors by candesartan, had no significant effect on J_V. In the present study, we found that basolateral lisinopril had no significant effect on J_V at a concentration of either 60 nM (filled triangle in Fig. 3B) or 240 nM (filled diamonds in Fig. 3B). Luminal lisinopril also did not have an effect on J_V, at either 60 nM (Fig. 1B) or 240 nM (filled circles in Fig. 2B). Only 100 µM luminal lisinopril had a statistically significant effect on J_V, and then only at 20% CO₂.

Because basolateral lisinopril substantially reduced J at 20% CO₂ (Fig. 3A) but did not have a significant effect J_V (Fig. 3B), the drug must have substantially increased the reabsorption rate for solutes other than NaHCO₃ (J_Other). In other words, reciprocal effects of lisinopril on J and J_Other stabilized fluid reabsorption at 20% CO₂.

J_Other. In Fig. 6A, the open circles and solid lines, the control J, summarize both the historical and present control data (n = 152) from Figs. 2A, 3A, and 4A. In Fig. 6C, we have similarly replotted the J data for basolateral 240 nM lisinopril (from Fig. 3A) and for luminal 100 µM lisinopril (from Fig. 4A). In Fig. 6, A and C, we also have plotted values for J_Other, which we computed (as described in the Fig. 6 legend) for each of these three sets of J and J_V data. In the absence of lisinopril (Fig. 6A), J_Other and J had a reciprocal relationship, which explains the relative stability of J_V in Figs. 2B, 3B, and 4B. Luminal lisinopril (100 µM) caused J_Other at 20% CO₂ (Fig. 6C) to rise significantly above the corresponding control values (Fig. 6A). The effects of 240 nM basolateral and 100 µM luminal lisinopril at 5% CO₂, as well as 240 nM basolateral lisinopril at 20% CO₂, on J_Other did not reach statistical significance.

Concentration of NaHCO₃ and other solutes in the reabsorbate. In Fig. 6B, we show the values that we computed for concentrations of NaHCO₃ ([NaHCO₃]) and "other" solutes ([Other]) in the PT reabsorbate under control conditions. In Fig. 6D, we show the comparable reabsorbate [NaHCO₃] and [Other] values for experiments with basolateral 240 nM lisinopril and luminal 100 µM lisinopril. Compared with the control values in Fig. 6B, basolateral 240 nM lisinopril caused [Other] to rise significantly at 20% CO₂, and luminal 100 µM lisinopril caused [Other] to rise significantly at 5 and 20% CO₂. The effects are just the opposite for [NaHCO₃]. Thus the tendency is for lisinopril to cause reciprocal changes in [Other] and [NaHCO₃], just as it tends to cause reciprocal changes in J_Other and J.

Conclusions. Our data indicate that the isolated perfused rabbit S2 PT secretes predominantly preformed ANG II, as opposed to either angiotensinogen or ANG I. We propose that the conversion of angiotensinogen to ANG I, as well as the subsequent conversion of ANG I to ANG II, takes place in an intracellular vesicle (Fig. 7). We suggest that the PT also secretes remnant angiotensinogen (which would presumably cross-react with angiotensinogen antibodies) and the NH₂-terminal dipeptide cleaved from ANG I, or breakdown products thereof.

The lumen of the ACE-containing vesicle is presumably readily accessible to basolateral but not to luminal lisinopril. At extremely high levels (e.g., 100 µM), luminal lisinopril could reach ACE-containing vesicles by either leaking across the apical membrane or passing first through tight junctions and then leaking across the basolateral membrane. Basolateral membranes tend to have higher permeabilities than do apical membranes (reviewed in Ref. 30), specific examples being the permeabilities to NH₃ (25, 27) and CO₂ (27).

In retrospect, it is perhaps not surprising that the conversion from angiotensinogen to ANG II should take place almost entirely within an intracellular vesicle, inasmuch as the contents of the vesicle would have protracted exposure, before the vesicle fuses with the apical membrane, to renin and ACE.

	GRANTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was funded by National Institute of Diabetes and Digestive and Kidney Diseases Program Project Grant P01 DK-17433.

	ACKNOWLEDGMENTS

 
We thank Duncan Wong for computer support.

	FOOTNOTES

 

Address for reprint requests and other correspondence: W. F. Boron, Dept. of Physiology and Biophysics, Case Western Reserve Univ., 10900 Euclid Ave., Cleveland, OH 44106-4970 (e-mail: walter.boron{at}case.edu)

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.

	REFERENCES

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

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