Mechanisms of HCOminus 3 secretion in the rabbit connecting segment

Shuichi Tsuruoka and George J. Schwartz

Department of Pediatrics, University of Rochester School of Medicine, Rochester, New York 14642


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The connecting tubule (CNT) contains alpha -(H+-secreting) and beta -(HCO-3-secreting) intercalated cells and is therefore likely to contribute to acid-base homeostasis. To characterize the mechanisms of HCO-3 transport in the rabbit CNT, in which there is little definitive data presently available, we microdissected the segments from the superficial cortical labyrinth, perfused them in vitro, measured net HCO-3 transport (JHCO-3) by microcalorimetry, and examined the effects of several experimental maneuvers. Mean ± SE basal JHCO-3 was -3.4 ± 0.1 pmol · min-1 · mm-1 (net HCO-3 secretion), and transepithelial voltage was -13 ± 1 mV (n = 47). Net HCO-3 secretion was markedly inhibited by removal of luminal Cl- or application of basolateral H+-ATPase inhibitors (bafilomycin or concanamycin), maneuvers that inhibit beta -intercalated cell function. Net HCO-3 secretion was not affected by inhibitors of alpha -intercalated cell function (basolateral Cl- removal, basolateral DIDS, or luminal H+-ATPase inhibitors). Net HCO-3 secretion was stimulated by isoproterenol and inhibited by acetazolamide. These data indicate that 1) CNTs secrete HCO-3 via an apical DIDS-insensitive Cl-/HCO-3 exchanger, mediated by a basolateral bafilomycin- and concanamycin-sensitive H+-ATPase; 2) inhibition of cytosolic carbonic anhydrase decreases HCO-3 secretion; and 3) stimulation of beta -adrenergic receptors increases HCO-3 secretion. The failure to influence net HCO-3 transport by inhibiting alpha -intercalated cell apical H+-ATPases or basolateral Cl-/HCO-3 exchange suggests that the CNT has fewer functioning alpha -intercalated cells than the cortical collecting duct. These are the first studies to examine the rate and mechanisms of HCO-3 secretion by the rabbit CNT; this is clearly an important segment in mediating acid-base homeostasis.

intercalated cell; distal nephron; collecting duct; hydrogen-adenosine 5'-triphosphatase; carbonic anhydrase; chloride/bicarbonate exchange; isoproterenol; microperfusion in vitro; bafilomycin; concanamycin; acetazolamide; 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; Sch-28080


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE CONNECTING TUBULE (CNT) joins distal tubules to the cortical collecting duct (CCD) (16). The CNT contains two types of cells: 1) the CNT cell and 2) the intercalated cell. The ratio of CNT cells to intercalated cells is ~5:4 (16). With this relatively large number of intercalated cells, it is likely that the CNT plays an important role in acid-base homeostasis.

There are two major types of intercalated cells: 1) alpha -intercalated cells secrete H+, and 2) beta -intercalated cells secrete HCO-3. Therefore, the relative distribution of intercalated cell subtypes in the rabbit CNT may determine the overall direction of acid-base transport. A morphological characterization of the CNT by Verlander et al. (43) using colocalization of carbonic anhydrase II and band 3 (anion exchanger AE1) to identify alpha -intercalated cells and carbonic anhydrase II and peanut lectin to identify beta -intercalated cells showed that ~50% of the intercalated cells in the CNT were alpha -intercalated cells. On the other hand, Muto et al. (27) published a microelectrode study using luminal reduction of chloride to hyperpolarize beta -intercalated cells, but not alpha -cells, and showed that 97% of intercalated cells were beta -type. Using a monoclonal antibody (B63) directed against the apical membrane of peanut lectin agglutinin-selected cells of the rabbit kidney cortex, Fejes-Toth et al. (9) found substantial labeling of beta -intercalated cells in both the CNT and CCD.

Several studies have compared other functions of the CNT with those of the CCD. Imai (15) showed that the transepithelial voltage of the CNT was -27.0 ± 2.7 mV compared with -3.5 ± 2.1 mV in the CCD. Also, the voltage response to isoproterenol is at least an order of magnitude more sensitive in the CNT. Compared with the CCD (32), the rate of Na+ absorption is approximately three to four times larger in the CNT (1), and that of K+ secretion is at least as large in the CNT (17). Thus the CNT appears to play a major role in the renal handling of several electrolytes.

In view of our interest in the types of functional intercalated cell types present in this segment, we chose to measure net HCO-3 transport in isolated perfused CNTs. We also attempted to characterize the mechanisms of HCO-3 transport and the relative contributions of alpha - and beta -intercalated cells to the overall HCO-3 flux. Lastly, we tested whether beta -adrenergic agents stimulate HCO-3 secretion in this segment.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Female New Zealand White rabbits weighing 1.8-2.8 kg and maintained on normal laboratory chow (Purina lab diet 5326; Purina Mills, Richmond, IN) plus free access to tap water were used. The animals were killed by intracardiac injection of 130 mg pentobarbital sodium after premedication with ketamine (44 mg/kg) and xylazine (5 mg/kg).

Tubule isolation and microperfusion. Kidneys were removed, and 1- to 2-mm coronal slices were made and transferred to chilled dissection medium containing (in mM) 145 NaCl, 2.5 K2HPO4, 2 CaCl2, 1.2 MgSO4, 5.5 D-glucose, 1 trisodium citrate, 4 sodium lactate, and 6 L-alanine, pH 7.4, 290 ± 2 mosmol/kgH2O (41, 42). CNT segments were microdissected from the superficial cortical labyrinth, because of the infrequent branching of the superficial compared with the deep cortical CNT (15).

In vitro microperfusion was performed according to the method of Burg and Green (5) with modifications (40-42). An isolated CNT was rapidly transferred to a 1.2-ml temperature- and environmentally controlled chamber mounted on an inverted microscope and perfused and bathed at 37°C with Burg's solution containing (in mM) 120 NaCl, 25 NaHCO3, 2.5 K2HPO4, 2 CaCl2, 1.2 MgSO4, 5.5 D-glucose, 1 trisodium citrate, 4 sodium lactate, and 6 L-alanine, 290 ± 2 mosmol/kg H2O, and gassed with 94% O2-6% CO2, yielding a pH 7.4 at 37°C (40-42). The specimen chamber was continually suffused with 94% O2-6% CO2 to maintain pH at 7.4 (34).

The collecting end of the segment was sealed into a holding pipette using Sylgard 184 (Dow Corning, Midland, MI). The perfused length of each segment was measured using an eyepiece micrometer. Fourteen nanoliter samples of tubular fluid were collected under water-saturated mineral oil by timed filling of a calibrated volumetric pipette. Collections were generally made in triplicate for each experimental period. The bathing solution was exchanged by a peristaltic pump at a rate of 14 ml/h to maintain constant solute concentrations.

HCO-3 transport. The concentration of total CO2 (assumed to be equal to that of HCO-3) in the perfusate (C0) and collected fluid (CL) was measured by microcalorimetry (Picapnotherm; Microanalytical Instrumentation, Mountain View, CA). Because there is no net water absorption in the CNT (15, 17, 31, 37) the rate of HCO-3 transport (JHCO-3) was calculated as JHCO-3 = (C0 - CL) × (VL/L), where VL is the rate of collection of tubular fluid (~1.5 nl/min), L is tubular length (in mm), and J is in pmol · min-1 · mm-1 tubular length. When JHCO-3 is more than 0 there is net HCO-3 absorption, when JHCO-3 is less than 0 there is net HCO-3 secretion. The sensitivity of the Picapnotherm was 10-20 counts/pmol HCO-3, so that for samples of 14.5 nl, there were 145-290 counts/mM. The coefficient of variation for a 20 mM standard measured in quadruplicate was <1% (<45 counts/sample of 4,500 counts). This level of sensitivity allowed us to reliably detect differences of 1 mM HCO-3 between perfused and collected fluids. In practice, we perfused tubules at ~1.5 nl/min, which corresponded to a flow rate of 3.5-4 nl · min-1 · mm-1 and generally resulted in a difference of ~1 mM between perfused and collected fluids under basal conditions. Each sample was determined in the Picapnotherm immediately after collection. The perfusate HCO-3 concentration, measured at the beginning and end of each experiment, averaged 24.4 mM.

Transepithelial voltage. Transepithelial voltage (Vte) was measured using the perfusion pipette as an electrode. The voltage difference between calomel cells connected via 3 M KCl agar bridges to perfusing and bathing solutions was measured with a high-impedance electrometer (World Precision Instruments, Sarasota, FL). Collections of tubular fluid were initiated once the Vte had stabilized (45-60 min), and readings were recorded at the conclusion of each collection.

Viability. Evidence for damaged cells and gross leak of perfusate was continually assessed by the inclusion of 0.15 mg/ml Fast green dye (Sigma) to each perfusate during the study (40, 41). The experiment was discarded if tubular damage was detected.

Experimental protocols. Net HCO-3 flux was first measured under basal conditions (Burg's solution in lumen and bath) and then after removal of Cl- or another experimental maneuver. Calcium concentration was raised to 6 mM in chloride-free solutions to allow for additional complexing by the substitute gluconate (37, 40, 41).

We used 5 nM bafilomycin A1 or 10 nM concanamycin to inhibit H+-ATPase (6, 23, 40, 41), 10 µM Sch-28080 {2-methyl-8-(phenylmethoxy) imidazo[1,2-alpha ]pyridine-3-acetonitrile} to inhibit gastric-type H+,K+-ATPase (11, 40, 48) (kindly provided by Dr. T. Sybertz, Schering-Plough Research Institute, Kenilworth, NJ), 50 µM DIDS to inhibit the basolateral Cl-/HCO-3 exchanger of alpha -intercalated cells (33, 40, 45), 1 µM isoproterenol to stimulate the apical Cl-/HCO-3 exchanger and HCO-3 secretion of beta -intercalated cells (12, 30), and 10 µM acetazolamide to inhibit cytosolic carbonic anhydrase II (20, 26, 35). The Cl- replacement and each inhibitor have previously been found to be reversible (15, 26, 34, 40, 41), and there was no change in HCO-3 transport over the time period of these experiments (data not shown).

Chemicals. Bafilomycin A1, DIDS, isoproterenol, and acetazolamide were purchased from Sigma Chemical (St. Louis, MO); concanamycin was purchased from Fluka Biochemical (Ronkonkoma, NY). All other reagents and chemicals were of analytic grade and purchased from Sigma. Each agent was dissolved in DMSO at 0.1% final concentration.

Analysis and statistics. Data are presented as means ± SE; n is the number of animals studied. Paired comparisons for each tubule were analyzed by paired t-test, and comparisons between tubules from control vs. other segments were analyzed by unpaired t-tests and one-way ANOVA plus the Tukey-Kramer or Bonferroni tests for multiple comparisons. Statistical software was used (Number Cruncher Statistical Software, Kaysville, UT). Significance was asserted when P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Baseline data. Net HCO-3 secretion was observed in all 47 CNT segments (Fig. 1, Table 1). The mean net HCO-3 flux was -3.38 ± 0.09 pmol · min-1 · mm-1, not significantly different from -3.87 ± 0.23 pmol · min-1 · mm-1 observed recently in 29 control CCDs (40). Both CNT and CCD were significantly different from our previously published results in outer medullary collecting ducts from the inner stripe, which absorbed net HCO-3 at a rate of 12.8 ± 0.3 pmol · min-1 · mm-1 (n = 25) (41).


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Fig. 1.   Net HCO-3 flux (JHCO3) and transepithelial voltage in connecting tubule (CNT) segments (open bars, n = 47) compared with previous data from cortical collecting duct (CCD) segments (hatched bars) (42) and outer medullary collecting duct (OMCD) segments from the inner stripe (cross-hatched bars) (43). a Significantly different from CCD (P < 0.05). b Significantly different from OMCD.


                              
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Table 1.   Mean transport data for experimental maneuvers

Vte differed among these three distal segments. The mean Vte of the CNT was -13.0 ± 0.2 mV, significantly different from that of the CCD (-4.1 ± 0.2 mV) or outer medullary collecting duct (+3.6 ± 0.2 mV).

Effect of luminal Cl- removal. To test the Cl- dependence of HCO-3 secretion in perfused CNT segments, we replaced all luminal Cl- by gluconate (Fig. 2, Table 1). This maneuver would presumably inhibit the secretion of HCO-3 in exchange for Cl- in beta -intercalated cells. The secretion of HCO-3 was markedly inhibited in the absence of luminal Cl- (-3.52 ± 0.15 pmol · min-1 · mm-1 to -0.17 ± 0.04, n = 5, P < 0.001), and there was no evidence for underlying net H+ secretion (positive HCO-3 flux) by alpha -intercalated cells. Associated with the loss of HCO-3 secretion was a decrease in collected fluid HCO-3 concentration from 25.5 ± 0.1 mM to virtually the perfusate concentration of 24.3 ± 0.1 mM. Allowing for the generation of a liquid junction potential of 6.8 mV (38), there was no significant change in Vte with Cl- removal (-12.7 ± 0.7 mV to -12.3 ± 0.6). This result was consistent with electroneutral HCO-3 secretion mediated by beta -intercalated cells in the CNT.


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Fig. 2.   Net HCO-3 flux in CNT segments under basal conditions and after removal of luminal Cl-. Each experiment is represented by a line without symbols (n = 5). * Significantly different from basal period by paired t-test (P < 0.05).

Effect of bath Cl- removal. We next tested whether inhibition of basolateral anion exchange, which is normally active in alpha -intercalated cells, would have any effect on net HCO-3 flux (Fig. 3, Table 1). There was no significant change in the rate of HCO-3 secretion (-3.41 ± 0.22 pmol · min-1 · mm-1 to -3.43 ± 0.23, n = 5) or collected fluid HCO-3 concentration (25.4 ± 0.1 mM to 25.4 ± 0.1) with bath Cl- removal; however there was a small significant decrease in Vte (-11.7 ± 0.6 mV to -11.4 ± 0.6). The relative magnitude of the increase in HCO-3 secretion, as alpha -intercalated cell H+ secretion was presumably inhibited by bath Cl- removal, was <1%. The results of this protocol suggested that countervailing H+ secretion by alpha -intercalated cells was not detectable.


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Fig. 3.   Net HCO-3 flux in CNT segments under basal conditions and after removal of Cl- from the bath. Each experiment is represented by a line without symbols (n = 5).

Effect of luminal DIDS. We used 50 µM of the stilbene DIDS to try and inhibit the apical anion exchanger of beta -intercalated cells (Table 1), realizing that this agent does not inhibit HCO-3 secretion by CCD beta -intercalated cells (30). Indeed, DIDS in the lumen did not significantly change net HCO-3 flux (-3.82 ± 0.22 pmol · min-1 · mm-1 to -3.82 ± 0.23, n = 4) or Vte (-12.8 ± 0.6 mV to -12.8 ± 0.6). There was a small decrease in collected fluid HCO-3 concentration (25.5 ± 0.1 mM to 25.3 ± 0.1, P < 0.01) that was probably associated with a minimal increase in flow rate. The results of this protocol suggested that the apical anion exchanger in beta -intercalated cells was DIDS-insensitive in the CNT, as in the CCD.

Effect of bath DIDS. To further examine the role of alpha -intercalated cells in contributing to net HCO-3 transport, we inhibited the basolateral anion exchanger AE1 with 50 µM DIDS (Table 1). Indeed, bath DIDS had no significant effect on net HCO-3 flux (-2.61 ± 0.23 pmol · min-1 · mm-1 to -2.66 ± 0.31, n = 6), collected fluid HCO-3 concentration (25.1 ± 0.1 mM to 25.1 ± 0.2), or Vte (-13.7 ± 0.6 mV to -13.0 ± 0.7). The relative magnitude of the increase in HCO-3 secretion, as alpha -intercalated cell H+ secretion was inhibited by DIDS, was only 2% and not significant. These results suggested that alpha -intercalated cells did not contribute substantially to the overall net HCO-3 flux.

Effect of luminal bafilomycin. By inhibiting the luminal H+-ATPase with bafilomycin, we were able to further assess the contribution of alpha -intercalated cells to net HCO-3 transport (Table 1). In keeping with the previous studies showing little effect of inhibiting alpha -intercalated cell function, luminal bafilomycin caused a small (1%) but significant increase in net HCO-3 secretion (-3.91 ± 0.27 pmol · min-1 · mm-1 to -3.95 ± 0.27, n = 4, P < 0.05), and no change in Vte (-14.4 ± 0.3 mV to -14.3 ± 0.3). There was a minimal decrease in collected fluid HCO-3 concentration (25.5 ± 0.1 mM to 25.4 ± 0.2, P < 0.01), which was also associated with a comparably lower perfusate HCO-3 concentration (24.4 ± 0.1 mM vs. 24.2 ± 0.2, P < 0.05). These data did not suggest a major contribution of alpha -intercalated cells to mediating HCO-3 transport in the CNT.

Effect of bath bafilomycin and concanamycin. It is believed that beta -intercalated cells have a basolateral H+-ATPase to generate HCO-3 for secretion into the luminal fluid. If this pump were inhibited by 5 nM bafilomycin, then net HCO-3 secretion by beta -intercalated cells would also be inhibited (Fig. 4, Table 1). Indeed, bath bafilomycin completely inhibited net HCO-3 secretion (-3.63 ± 0.34 pmol · min-1 · mm-1 to -0.14 ± 0.05, n = 5, P < 0.001), reduced collected fluid HCO-3 concentration to the level of the perfusate concentration (25.5 ± 0.1 mM to 24.3 ± 0.1, P < 0.01) and diminished the Vte (-13.3 ± 1.1 mV to -5.0 ± 1.4, P < 0.001). The inhibition of the electrogenic basolateral H+-ATPase would be expected to reduce luminal electronegativity in addition to obliterating HCO-3 secretion.


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Fig. 4.   Net HCO-3 flux in CNT segments under basal conditions and after addition of 5 nM bafilomycin to the bath. Each experiment is represented by a line without symbols (n = 5). * Significantly different from basal period by paired t-test (P < 0.05).

We confirmed these findings by using a different H+-ATPase inhibitor, concanamycin, at 10 nM (Fig. 5, Table 1). This agent also markedly inhibited net HCO-3 secretion (-3.07 ± 0.1 pmol · min-1 · mm-1 to -0.13 ± 0.02, n = 3, P < 0.001), reduced collected HCO-3 concentration (25.5 ± 0.1 mM to 24.4 ± 0.1, P < 0.01), and Vte (-12.0 ± 0.4 mV to -4.1 ± 0.4, P < 0.001). These results showed that HCO-3 secretion was inhibited by basolateral H+-ATPase inhibitors, suggesting that beta -intercalated cells with basolateral H+ pumps were mediating this flux.


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Fig. 5.   Net HCO-3 flux in CNT segments under basal conditions and after addition of 10 nM concanamycin to the bath. Each experiment is represented by a line without symbols (n = 3). * Significantly different from basal period by paired t-test (P < 0.05).

Effect of luminal Sch-28080. It is believed that beta -intercalated cells have an apical gastric-like H+-K+-ATPase that may also mediate H+ transport and would be sensitive to the inhibitor Sch-28080 at 10 µM (36, 46). If this pump were inhibited, then net HCO-3 secretion by beta -intercalated cells would be stimulated (Table 1). We found, however, that luminal Sch-28080 had no significant effect on net HCO-3 transport (-3.62 ± 0.32 pmol · min-1 · mm-1 to -3.64 ± 0.31, n = 5) or collected fluid HCO-3 concentration (25.3 ± 0.1 mM to 25.3 ± 0.1), and a minimal effect on Vte (-13.4 ± 0.7 mV to -13.2 ± 0.7, P < 0.05). These results showed that the apical H+-K+-ATPase did not play a major role in HCO-3 transport under baseline conditions in the CNT.

Effect of bath Sch-28080. The possibility that the H+-K+-ATPase might be located basolaterally in these intercalated cells was considered by adding 10 µM Sch-28080 to the bathing solution. In this case the inhibition of a basolateral H+-K+-ATPase would result in diminished HCO-3 secretion in perfused CNT segments (Table 1). We found that basolateral Sch-28080 had no effect on net HCO-3 transport (-3.25 ± 0.12 pmol · min-1 · mm-1 to -3.23 ± 0.15, n = 4), collected fluid HCO-3 concentration (25.4 ± 0.1 mM to 25.5 ± 0.1), or Vte (-12.5 ± 0.5 mV to -12.4 ± 0.5). These results showed that a putative basolateral H+-K+-ATPase did not play a major role in mediating net HCO-3 secretion in the CNT under baseline conditions.

Effect of bath isoproterenol. Data from the perfused CCD indicate that isoproterenol stimulates HCO-3 secretion and apical Cl-/HCO-3 exchange (12, 30). Also, isoproterenol has been found to decrease the lumen- negative voltage of the perfused CCD, perhaps by stimulating electrogenic Cl- transport out of the lumen (13). In addition, the Vte of the perfused CNT is more sensitive than the CCD to isoproterenol in the micromolar range (15). We therefore examined the effect of 1 µM isoproterenol added to the bathing solution on net HCO-3 transport in CNT segments (Fig. 6, Table 1). Isoproterenol caused a large decrease in lumen-negative Vte (-14.0 ± 0.6 mV to -7.8 ± 0.2, n = 6, P < 0.001), while also stimulating net HCO-3 secretion by 72% (-3.48 ± 0.21 pmol · min-1 · mm-1 to -5.98 ± 0.34, P < 0.001) and increasing collected fluid HCO-3 concentration by 0.7 mM (25.4 ± 0.1 mM to 26.1 ± 0.2, P < 0.01). These results indicated that isoproterenol significantly stimulated net HCO-3 secretion by beta -intercalated cells of the CNT, as in the CCD. Both segments appear to be comprised primarily of beta - rather than alpha -intercalated cells, at least by function; but the CNT, unlike the CCD (42), did not appear to show measurable alpha -cell function after inhibition of the beta -cells.


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Fig. 6.   Net HCO-3 flux in CNT segments under basal conditions and after addition of 1 µM isoproterenol to the bath. Each experiment is represented by a line without symbols (n = 6). * Significantly different from basal period by paired t-test (P < 0.05).

Effect of bath acetazolamide. The secretion of HCO-3 is known to be dependent on cytosolic carbonic anhydrase II, since inhibition of carbonic anhydrase with the permeant agent acetazolamide eliminates net HCO-3 secretion in perfused CCDs without affecting lumen-negative Vte (24). We examined the effect of adding 10 µM acetazolamide to the bath on net HCO-3 transport in perfused CNT segments (Fig. 7, Table 1); this lower concentration (24, 34, 35) was used to minimize nonspecific effects and yet inhibit most of the carbonic anhydrase. We found that acetazolamide inhibited 84% of net HCO-3 secretion (-3.42 ± 0.25 pmol · min-1 · mm-1 to -0.56 ± 0.10, n = 5, P < 0.001), reduced collected HCO-3 concentration (25.4 ± 0.1 mM to 24.6 ± 0.1, P < 0.01), and inhibited Vte (-13.1 ± 0.9 mV to -6.1 ± 1.0, P < 0.01). These results showed that HCO-3 secretion by the perfused CNT was dependent on cytosolic carbonic anhydrase, and inhibition of this enzyme also blocked the basolateral electrogenic H+ pump and thereby reduced luminal Vte.


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Fig. 7.   Net HCO-3 flux in CNT segments under basal conditions and after addition of 10 µM acetazolamide to the bath. Each experiment is represented by a line without symbols (n = 5). * Significantly different from basal period by paired t-test (P < 0.05).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our studies demonstrate that the CNT plays a major role in acid-base homeostasis. In addition to exhibiting higher rates of Na+ absorption (1) and K+ secretion (17) than the CCD, the CNT secretes HCO-3 at rates comparable to that of the contiguous CCD. This finding indicates that the CNT contributes HCO-3 to the alkaline urine of rabbits. Indeed, in the rabbit with 200,000 nephrons per kidney (16) and at least 1 mm of CNT segments per nephron in the cortical labyrinth, there would be ~1.4 µmol HCO-3 secreted per min or 2 mmol/day. The CNT may secrete more HCO-3 than the CCD, which averages a similar transport rate over 3-4 mm of length but drains an average of six nephrons (16). Thus the CNT may be the most important HCO-3-secreting segment in the rabbit kidney.

We did not expect such a high rate of HCO-3 secretion in the CNT because of the relatively large numbers of band 3-positive intercalated cells found in the CNT by immunocytochemistry (43). Indeed, if 50% of the intercalated cells were alpha -type (band 3 positive) and 50% were beta -type (peanut lectin positive) (43), then the CNT segment would be expected to secrete HCO-3 or H+, depending on which intercalated cell function could be inhibited, much like the CCD (40).

The mechanisms of HCO-3 transport in the CNT have not been previously examined. Our data show (Table 2) that luminal Cl- removal or bath bafilomycin or bath concanamycin each markedly inhibited net HCO-3 secretion; acetazolamide inhibited nearly all HCO-3 secretion. These findings indicate that in the presence of cytosolic carbonic anhydrase II in beta -intercalated cells (3, 4, 18, 22, 28), cell water and CO2 generate carbonic acid, which dissociates to H+ and HCO-3. Protons are secreted across the basolateral membrane via a bafilomycin-sensitive vacuolar H+-ATPase, and HCO-3 is secreted by a Cl--dependent process, presumably Cl-/HCO-3 exchange, across the apical membrane. Evidence for the electrogenic secretion of H+ across the basolateral membrane in beta -intercalated cells was noted in the reduction in electronegativity of Vte with inhibitors of the H+-ATPase or of H+ pumping. There was no demonstrable effect on HCO-3 transport or Vte with luminal DIDS, suggesting that this apical Cl-/HCO-3 exchanger was not DIDS sensitive, as has been established for the apical anion exchanger of CCD beta -intercalated cells (9, 30).

                              
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Table 2.   Effect of inhibitors on Delta JHCO-3 in CNT

These findings are compatible with the microelectrode studies of Muto et al. (27), which demonstrated functionally that 97% of the intercalated cells were of the beta -type. We would assume that a failure to observe net H+ secretion (HCO-3 absorption) during inhibition of HCO-3 secretion (Tables 1 and 2) is consistent with a lack of many functioning alpha -intercalated cells. Clearly, the histological finding of band 3-labeled cells in the CNT would suggest otherwise; immunocytochemical identification of subunits of the vacuolar H+-ATPase in the CNT is certainly required. Perhaps, these "alpha -intercalated cells" do not express the H+ pumps required for net HCO-3 absorption. Furthermore, the lack of effect of luminal bafilomycin, a specific H+-ATPase inhibitor at nanomolar concentrations (6, 23), tends to rule out a significant contribution to the net HCO-3 flux from apical H+ pumps. The lack of effect of basolateral DIDS, an inhibitor of band 3, also suggests minimal H+ secretion from the alpha -intercalated cells. It is possible that these cells secrete protons not via a vacuolar H+-ATPase but rather via a gastric-type H+-K+-ATPase (36, 44, 46). The failure of Sch-28080 to affect net HCO-3 flux suggested that this type of H+-K+-ATPase does not contribute significantly to the net transport of HCO-3.

The removal of Cl- from the bath would be expected to inhibit basolateral Cl-/HCO-3 exchange, presumably of alpha -intercalated cells. Inasmuch as no effect of bath Cl- removal, bath DIDS, or luminal bafilomycin was noted (see Table 2), the contribution of alpha -intercalated cells was probably small. Although both types of intercalated cells are believed to have basolateral Cl- conductances (10, 27), the removal of bath Cl- did not affect net HCO-3 transport. If alpha -intercalated cells were not contributing much to net HCO-3 secretion, then this maneuver to decrease Cl--dependent HCO-3 efflux across the basolateral membrane of these cells would not be expected to have much effect. Regarding beta -intercalated cells in the CCD, the removal of bath Cl- would cause a decrease in cell Cl-, which enhances apical Cl-/HCO-3 exchange and therefore net HCO-3 secretion (25, 47); the increase in HCO-3 secretion then causes a loss of cellular base and a decrease in cell pH (29), which would then stimulate the basolateral H+ pump. Such a stimulation in net HCO-3 secretion was not observed in the CNT, despite a detectable basolateral Cl- conductance (27), suggesting that the apical Cl-/HCO-3 exchanger might have been operating at close to maximal capacity, or the Cl- conductances were small, or cell Cl- was already very low and not greatly affected by this maneuver. In addition, the use of DIDS, which blocks Cl- channels (21) in addition to its inhibition of band 3-like Cl-/HCO-3 exchangers, failed to affect net HCO-3 flux. Measurements of the DIDS- and Cl--induced changes in intercalated cell pH in perfused CNT segments might help us to better understand the role of Cl- in mediating HCO-3 secretion.

An examination of the CCD's response to isoproterenol provides further information about the mechanism of HCO-3 secretion in the CNT. It is well known that isoproterenol and other beta -agonists increase cAMP formation in beta -intercalated cells (8). Schuster (30) found that 1 µM isoproterenol in the bath increased CCD HCO-3 secretion by 44% and depolarized the Vte by 85%. There was also a significant correlation between baseline HCO-3 secretory rates and the increments in HCO-3 secretion induced by isoproterenol (30). Iino et al. (13) showed that isoproterenol decreased Vte by 51% but did not affect isotopic lumen-to-bath Na+ flux; however, isoproterenol stimulated Cl- absorption by 145%. Interestingly, acetazolamide abolished the effect of isoproterenol on Vte, suggesting that the beta -agonist was stimulating some form of Cl- absorption that depended on H+/HCO-3 transport. Kimmel and Goldfarb (19) found that 1 µM isoproterenol caused K+ secretion to decrease by 41% and Vte by 53% in the CCD and that removing Cl- from the bath and perfusate attenuated the ability of isoproterenol to reduce K+ secretion and Vte. Fluorescent cell pH studies indicate that isoproterenol causes a decrease in pH of beta -intercalated cells (7, 12), and the pH change was prevented if Cl- was removed from the luminal fluid. Finally, isoproterenol stimulates Cl-/Cl- self-exchange (39). Each of these results would indicate that the apical Cl-/HCO-3 exchanger is required for the action of isoproterenol. Thus isoproterenol stimulates HCO-3 secretion and Cl- absorption via Cl-/HCO-3 exchange in beta -intercalated cells of the CCD. The mechanism for the depolarization of Vte may become apparent in the analysis of induced Cl- conductances.

In the turtle bladder, cAMP stimulates HCO-3 secretion along with a reversal of the Vte (from serosa negative to positive) (39). These authors believed that cAMP may induce electrogenic HCO-3 secretion by opening an apical Cl- (also HCO-3) conductance. If the induced apical anion channel allowed the passage of HCO-3, in addition to the electroneutral transport via the apical Cl-/HCO-3 exchanger, then there would be electrogenic HCO-3 secretion.

If such an apical anion channel were induced in the nephron segment by isoproterenol, via elevating cell cAMP (2, 14), then the Vte would tend to become more negative with anion entry into the lumen. This has not been observed in the CCD (13, 15, 19, 30) or in the CNT (15); rather, the Vte becomes depolarized (less negative) after isoproterenol is added to the bath. Our studies in the CNT confirm this observation.

Perhaps the change in Vte in response to isoproterenol may be better understood in the context of increasing Cl- absorption. If the putative apical Cl- conductance were induced by isoproterenol, then there would be, in addition to increased electroneutral Cl- absorption, increased electrogenic Cl- absorption, as shown by Iino et al. (13): the flow of negative charges out of the lumen would depolarize Vte as observed by us and others (13, 15, 19, 30). On the other hand, if a basolateral Cl- conductance were activated by cAMP-isoproterenol in beta -intercalated cells, then there would be increased apical Cl- entry via Cl-/HCO-3 exchange due to the increased driving force for intracellular Cl-. This would result in increased electroneutral HCO-3 secretion and Cl- absorption, probably with little change in Vte, which was not observed in the CNT. Thus, in response to isoproterenol, the induction of an apical Cl- conductance allowing for electrogenic Cl- absorption, in addition to increased electroneutral HCO-3 secretion and Cl- absorption, appears to be required to explain the findings observed in the CCD and CNT. The additional opening of the basolateral Cl- conductance would further help to stimulate the transcellular fluxes of Cl- and possibly HCO-3 and could therefore contribute to the luminal depolarization, as well as to the increase in HCO-3 secretion and Cl- absorption.

In summary, we have shown that the CNT segment plays a major role in acid-base homeostasis. This segment secretes HCO-3 at rates comparable to that of the contiguous CCD. The secretion of HCO-3 requires apical Cl-, indicating the probability that it is mediated by apical Cl-/HCO-3 exchange. It also requires cytosolic carbonic anhydrase. The pump driving HCO-3 secretion appears to be a basolaterally located H+-ATPase. From our functional studies we conclude that these are beta -intercalated cells. The contribution of alpha -intercalated cells to baseline HCO-3 transport appears to be small, because net transport was not substantially influenced by luminal H+-ATPase inhibitors or inhibition of basolateral Cl-/HCO-3 exchange. These studies indicate that the CNT should be included with the collecting duct segments as important contributors to renal acid-base homeostasis and could be quantitatively the most important HCO-3-secreting segment in the kidney.


    ACKNOWLEDGEMENTS

We are grateful for the technical assistance of A. Kittelberger and D. Barnhart.


    FOOTNOTES

S. Tsuruoka was supported by a postdoctoral fellowship award from the American Heart Association, New York State Affiliate. G. J. Schwartz was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-50603.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: G. J. Schwartz, Div. of Pediatric Nephrology, Dept. of Pediatrics, PO Box 777, Univ. of Rochester School of Medicine, 601 Elmwood Ave., Rochester, NY 14642 (E-mail: George_Schwartz{at}URMC.Rochester.edu).

Received 12 February 1999; accepted in final form 26 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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