Abstract
The Cl−/HCO3− exchanger AE2 is believed to be involved in transcellular bicarbonate reabsorption that occurs in the thick ascending limb of Henle’s loop (TAL). The purpose of this study was to test whether chronic changes in acid-base status and sodium intake regulate AE2 polypeptide abundance in the TAL of the rat. Rats were subjected to 6 d of loading with NaCl, NH4Cl, NaHCO3, KCl, or KHCO3. AE2 protein abundance was estimated by semiquantitative immunoblotting in renal membrane fractions isolated from the cortex and the outer medulla of treated and control rats. In the renal cortex, AE2 abundance was markedly increased in response to oral loading with NH4Cl or with NaCl. In contrast, AE2 abundance was unchanged in response to loading with KCl or with NaHCO3 and was decreased by loading with KHCO3. The response of AE2 in the outer medulla differed from that in the cortex in that HCO3− loading increased AE2 abundance when administered with Na+ but had no effect when administered with K+. Immunohistochemistry revealed that NaCl loading increased AE2 abundance in the basolateral membrane of both the cortical and the medullary TAL. In contrast, NH4Cl loading increased AE2 abundance only in the cortical TAL but not in the medullary TAL. These results suggest that regulation of the basolateral Cl−/HCO3− exchanger AE2 plays an important role in the adaptation of bicarbonate absorption in the TAL during chronic acid-base disturbances and high sodium intake. The present study also emphasizes the contribution of cortical TAL adaptation in the renal regulation of acid-base status.
The kidney controls systemic acid-base balance through the coordinated regulation of multiple transport processes along the length of the nephron that together allow adaptation of HCO3− reabsorption [for review, see ref (1)]. Eighty-five percent of filtered bicarbonate is reabsorbed by the proximal tubule (PT), and the thick ascending limbs reabsorb the remaining 15%. In response to metabolic acidosis, HCO3− reabsorption by PT cells is stimulated by an increase in expression and activity of the apical Na+/H+ exchanger NHE3, in conjunction with increased activity of the basolateral Na+-(HCO3−)n co-transporter kNBCe1 (2–4⇓⇓). Conversely, chronic metabolic alkalosis leads to the opposite regulation (5,6⇓). Although bicarbonate absorption occurs along the entire course of the thick ascending limb (TAL), its regulation has been studied most extensively in the medullary part of the TAL (MTAL). As in PT, chronic metabolic acidosis is associated with adaptive increases in MTAL bicarbonate reabsorption and enhanced apical NHE3 activity and expression (2,7,8⇓⇓). The MTAL response to chronic metabolic alkalosis depends on the experimental model. In rats with chronic chloride depletion metabolic alkalosis with unaltered sodium intake, the HCO3− absorptive capacity of the MTAL is reduced (9). However, in rats with chronic metabolic alkalosis induced by chronic ingestion of NaHCO3, the HCO3− absorptive capacity of the MTAL is paradoxically enhanced (7), despite appropriate downregulation of apical NHE3 (10). The increased HCO3− absorption that follows NaHCO3 loading has been hypothesized to reflect adaptation of the MTAL to high sodium intake, an important determinant of MTAL bicarbonate absorption (7).
We have previously shown that the major basolateral HCO3− exit pathway of MTAL cells is via Cl−/HCO3− exchange activity most plausibly attributed to AE2 (11–13⇓⇓). AE2 is also expressed in the cortical portion of the TAL (11). It thus is possible that regulation of AE2 expression is involved in adaptive change in TAL HCO3− reabsorption during acid-base disorders and alterations in sodium intake. Therefore, the present study compared changes in renal AE2 polypeptide abundance in response to chronic oral loading with NH4Cl, NaHCO3, KHCO3, NaCl, and KCl.
Materials and Methods
Animal Treatments
Male Sprague-Dawley rats (IFFA-CREDO, L’Arbresle, France) that weighed 240 to 260 g were used for this study.
First Protocol (NH4Cl, NaHCO3 and NaCl Loading).
Five independent series were performed. In each series, a control group and three treated groups were handled in parallel over a 6-d period. Each group consisted of two rats. Treated rats were given 0.28 M NH4Cl, NaHCO3, or NaCl and were fed ad libitum with standard laboratory rat chow (Dietex, Saint-Gratien, France). Control rats were fed ad libitum with standard laboratory rat chow and had free access to distilled water for the same period of time.
Second Protocol (KCl and KHCO3 Loading).
Two independent series were performed. In each series, a control group and two treated groups were handled in parallel over a 6-d period. Each group consisted of three rats. Treated rats were given 0.28 M of either KCl or KHCO3 and were fed ad libitum with standard laboratory rat chow (Dietex). Control rats were fed ad libitum with standard laboratory rat chow and had free access to distilled water for the same period of time.
At the end of the experimental period, blood was collected by aortic puncture under anesthesia, provided by peritoneal injection of pentobarbital sodium. The bladder was punctured just after anesthesia, and urine was withdrawn. Kidneys were rapidly removed and immersed into ice-cold Hank’s modified solution that contained (in mM) 137 NaCl, 5.4 KCl, 25 NaHCO3, 0.3 Na2HPO4, 0.4 KH2PO4, 0.5 MgCl2, 10 HEPES, 5 glucose, 1 leucine, and 1 mg/ml BSA. Arterial pH, pCO2, and pO2 were measured with an AVL Compact 1 pH/blood-gas analyser (AVL Instruments Médicaux, Eragny-sur-Oise, France). Serum and urine electrolytes and creatinine were measured by standard methods with a Beckman LX20 autoanalyser (Coulter-Beckman, Villepinte, France) and Olympus AU 400 (Olympus, Rungis, France). Plasma aldosterone was measured by RIA (DPC Dade Behring, La Défense, France). Urinary ammonia was measured by the method of Berthelot (14). Urinary pH and HCO3− were measured with an AVL 555 Radiometer analyser (AVL Instruments Médicaux).
Preparation of Membrane Fractions
Excised kidneys were removed and cut into 5-mm slices. The cortex and the outer medulla (inner stripe) from the kidneys of two rats were excised under a stereoscopic microscope and placed into ice-cold isolation buffer (250 mM sucrose and 20 mM Tris-HEPES [pH 7.4]) that contained protease inhibitors (in μg/ml): 4 aprotinin, 4 leupeptin, 1.5 pepstatin A, and 28 4-(2-aminoethyl)-benzenesulfonyl fluoride. Minced tissues were homogenized in a Dounce homogenizer (pestle A × 5) followed by five passes through a Teflon-glass homogenizer rotating at 1000 rpm. The homogenate was centrifuged at 1000 × g for 10 min, and the supernatant was centrifuged at 100,000 × g for 20 min at 4°C. The pellet was resuspended in isolation buffer. Protein concentration was determined using the Bradford protein assay.
Antibodies
Rabbit polyclonal antibody to mouse C-terminal AE2 amino acids 1224 to 1237 has been previously characterized (11,13⇓). Affinity-purified rabbit polyclonal antibodies that recognize the rat and mouse AE2a and AE2b isoforms (anti-AE2a/b [aa 101 to 117]) as described previously by Frische et al. (15) were used for immunohistochemistry of rat kidney sections.
Immunoblot Analyses
Membrane proteins were solubilized in SDS-loading buffer (10 mM Tris HCl [pH 6.8], 1% SDS, 2% dithiothreitol, 13% glycerol, and bromophenol blue), incubated at 20°C for 30 min. Electrophoresis was initially performed for all samples on 7.5% polyacrylamide minigels (XCell SureLock Mini-cell; Invitrogen Life Technologies), which were stained with Coomassie blue to provide quantitative assessment of loading. Figure 1A shows one representative experiment of Coomassie blue–stained control gels. Densitometry analyses (Figure 1B) revealed no variation in the Coomassie blue band densities, indicating operationally identical protein loading of the different samples. For immunoblotting, proteins were transferred electrophoretically (XCell II Blot Module; Invitrogen Life Technologies) for 1.5 h at 4°C from unstained gels to nitrocellulose membranes (Amersham, Arlington Heights, IL) and then stained with 0.5% Ponceau S in acetic acid to check uniformity of protein transfer onto the nitrocellulose membrane. Posttransfer staining of gels with Coomassie blue confirmed complete protein transfer. Initial electrophoretic transfers used two membranes in tandem. The absence of transferred protein on the membrane placed farther away from the gel indicated that significant amounts of protein did not pass through the membrane to be probed. Membranes were first incubated in 5% nonfat dry milk in PBS (pH 7.4) for 1 h at room temperature to block nonspecific binding of antibody, followed by overnight at 4°C with the anti-AE2 (aa 1224 to 1237) antibody (diluted 1:300 for the cortical samples or 1:8000 for the medullary samples) in PBS that contained 1% nonfat dry milk. After four 5-min washes in PBS that contained 0.1% Tween-20, membranes were incubated with 1:3000 dilution of goat anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad, Hercules, CA) in PBS that contained 5% nonfat dry milk for 2 h at room temperature. Blots were washed as above, and luminol-enhanced chemiluminescence (NEN Life Science Products, Inc., Boston, MA) was used to visualize bound antibodies on Hyperfilm ECL (Amersham). The autoradiographs were digitized by laser scanner (Epson Perfection 1650; Epson), and bands were quantified using NIH Image software. Densitometric values were normalized to the mean for the control group that was defined as 100%, and results were expressed as mean ± SEM.
Figure 1. Representative Coomassie blue–stained polyacrylamide gels used to control protein loading. The gels presented here show aliquots of the same samples presented in Figure 2. (A) Control rats (c) and acidotic rats (a). (B) Densitometric analyses of total electrophoresed protein separated on these gels established that the accompanying immunoblots (loaded identically; Figure 2A) were indeed uniformly loaded. Bars represent means ± SEM.
Immunohistochemistry of Rat Kidney Sections
Kidneys from three NH4Cl-loaded rats, six NaCl-loaded rats, and nine control rats were fixed with 4% paraformaldehyde. Kidney slices that contained all kidney zones were dehydrated and embedded in paraffin. The paraffin-embedded tissues were cut at 2-μm thickness on a rotary microtome (Leica, Wetzlar, Germany), dewaxed in xylene, and rehydrated through 99% ethanol to 96% ethanol. At this hydration level, endogenous peroxidase activity was blocked by 30-min incubation in 0.3% H2O2 in methanol. Rehydration was completed through 96 and 70% ethanol. For revealing antigens, sections were placed in 10 mM Tris buffer (pH 9.0) that contained 0.5 mM EGTA (Titriplex VI, Merck) and heated in a microwave oven for 10 min. Nonspecific binding of Ig was prevented by incubating the sections in 50 mM NH4Cl for 30 min followed by blocking in PBS supplemented with 1% BSA, 0.05% saponin, and 0.2% gelatin. Sections were incubated overnight at 4°C with affinity-purified anti-AE2a/b (aa 101 to 117) antibodies diluted 1:4000 (for the NaCl and NaCl controls) or 1:8000 (for NH4Cl and NH4Cl controls) in 10 mM PBS (pH 7.4) that contained 0.1% Triton X-100 and 0.1% BSA, and for 2 h at room temperature with horseradish peroxidase–linked goat anti-rabbit secondary antibodies (P448; DAKO, Glostrup, Denmark). Labeling was visualized by 3,3-diaminobenzidine technique, and the sections were counterstained using Mayer’s hematoxylin.
Statistical Analyses
All data are presented as means ± SEM. Comparisons among groups were assessed by ANOVA with Bonferroni correction or unpaired t test. P < 0.05 was considered significant.
Results
AE2 Abundance Was Markedly Stimulated during Metabolic Acidosis in the Cortex and the Outer Medulla, Whereas Metabolic Alkalosis Induced by NaHCO3 Loading Increased AE2 Abundance Only in the Outer Medulla
To test whether chronic metabolic acidosis and alkalosis would change the abundance of AE2 protein in the rat kidney, we prepared semiquantitative immunoblots with membrane fractions from the renal cortex and outer medulla (inner stripe) of control rats and of rats that were subjected to chronic loading with NH4Cl or NaHCO3. Plasma electrolytes and acid-base status of the control and the NH4Cl- or NaHCO3-loaded groups are summarized in Table 1. Rats that were subjected to NH4Cl loading (5 to 9 mmol/d) developed metabolic acidosis. This was reflected in a significant decrease in blood HCO3− concentration from the control value of 27.7 ± 0.5 to 23.1 ± 1.3 mmol/L (P < 0.05) and in a fall of blood pH from the control value of 7.37 ± 0.01 to 7.27 ± 0.03 (P < 0.05). Conversely, rats that were subjected to NaHCO3 loading developed metabolic alkalosis with a significant increase in plasma HCO3− concentration to 31.2 ± 0.8 mmol/L (P < 0.05, versus control group values). As expected, acidotic rats exhibited higher urinary ammonium excretion than control rats (73 ± 10 versus 9 ± 2 mmol/mmol creatinine; P < 0.05), whereas urinary ammonium excretion in alkalotic rats decreased to 1 ± 0.5 mmol/mmol creatinine (P < 0.05).
Table 1. Functional data in rats after chronic NaCl, NaHCO3, and NH4Cl loadinga
Figure 2A shows the results of immunoblots that were probed with anti-AE2 antibody (aa 1224 to 1237). As shown in Figure 2 and Table 2, metabolic acidosis induced a large increase in AE2 abundance in the cortex (644 ± 37% in the acidosis group versus 100 ± 15% in the control group; P < 0.001) and the outer medulla (238 ± 45% in the acidosis group versus 100 ± 15% in the control group; P < 0.05). Semiquantitative immunoblotting of membrane fractions from the alkalotic rats showed that AE2 abundance was significantly increased in the outer medulla (176 ± 25% in the alkalosis group versus 100 ± 15% in the control group; P < 0.05) but not in the cortex (126 ± 21% versus 100 ± 10% in the control group; NS; Figure 3, Table 2).
Figure 2. Effects of chronic metabolic acidosis induced by NH4Cl loading on AE2 protein abundance in the rat renal kidney. (A) Immunoblots of membrane fractions from the cortex and outer medulla (inner stripe) obtained from five acidotic rats (ac) and their respective control rats (c). Each lane was loaded with 10 μg of protein. Immunoblots were reacted with anti-AE2 antibody and revealed the expected 180-kD band. (B) Densitometric analyses from five independent experiments revealed a significant increase in AE2 abundance in acidotic rats compared with control rats in the cortex and the outer medulla. Bars represent means ± SEM. *Statistically significant increase versus control (P < 0.05).
Table 2. Summary of densitometric analysis of immunoblotsa
Figure 3. Effects of chronic metabolic alkalosis induced by NaHCO3 loading on AE2 protein abundance in the rat kidney. (A) Immunoblots of membrane fractions from the cortex and outer medulla (inner stripe) obtained from five alkalotic rats (al) and their respective controls (c). Each lane was loaded with 10 μg of protein. Immunoblots were reacted with anti-AE2 antibody and revealed the expected 180-kD band. (B) Densitometric analyses from five independent experiments showing that the abundance of AE2 in alkalotic rats was significantly increased in the outer medulla but not in the cortex when compared with control rats. Bars represent means ± SEM. *Statistically significant increase versus control (P < 0.05).
AE2 Abundance Was Also Increased in Response to High NaCl Intake
As the effect of NaHCO3 loading on AE2 abundance could have been the result of the increase in sodium intake rather than the effect of increased bicarbonatemia, we also tested in parallel the effect on AE2 abundance of the same Na+ intake administered as NaCl loading. Table 1 shows functional data obtained in rats that were subjected to chronic NaCl loading. As expected, urinary sodium excretion was markedly increased in rats that were exposed to high sodium intake (NaCl or NaHCO3 loading) compared with control rats. Importantly, there was no difference between sodium excretion of NaCl-loaded rats compared with NaHCO3-loaded rats. In contrast to NaHCO3 loading, NaCl loading did not change blood pH, plasma HCO3−, and serum chloride concentrations when compared with control rats.
Figure 4 and Table 2 show that chronic NaCl loading markedly stimulated AE2 abundance in the outer medulla (202 ± 11% in NaCl-loaded rats versus 100 ± 13% in the control group; P < 0.001). In contrast to NaHCO3 loading, NaCl loading also increased AE2 abundance to the same extent in the cortex (201 ± 26% in NaCl-loaded rats versus 100 ± 7% in the control group; P < 0.01). Taken together, the results shown in Figures 3 and 4⇓ suggest that increased Na+ intake itself increases kidney AE2 abundance.
Figure 4. Effects of chronic sodium loading induced by NaCl loading on AE2 protein abundance in the rat kidney. (A) Immunoblots of membrane fractions from the cortex and the outer medulla (inner stripe) obtained from a control rat (c) and its pair-fed NaCl-loaded rat (na). Each lane was loaded with 10 μg of protein. Immunoblots were reacted with anti-AE2 antibody and revealed the expected 180-kD band. (B) Densitometric analyses from five independent experiments showing that the abundance of AE2 in alkalotic rats was significantly increased in the cortex and the outer medulla compared with control rats. Bars represent means ± SEM. *Statistically significant increase versus control (P < 0.05).
AE2 in the Renal Cortex Was Downregulated in Response to Chronic KHCO3 Loading
Because Na+ loadings experiments, administered with either Cl− or HCO3−, indicated that Na+ may exert an independent stimulatory effect on AE2, we hypothesized that the expected downregulation of AE2 in response to an increased bicarbonatemia may have been masked by an opposing upregulation as a result of the parallel increase in Na+ intake during the NaHCO3-loading protocol. To test this hypothesis, we next studied the effects of the same HCO3− load administered with K+ as the accompanying cation rather than Na+. Functional data are presented in Table 3. Rats that were subjected to KHCO3 loading developed metabolic alkalosis with an increase in plasma HCO3− concentration from the control value of 24.7 ± 0.9 to 29.3 ± 0.9 mmol/L in treated rats (P < 0.05) and exhibited a significantly higher urinary pH and bicarbonate excretion. Figure 5 and Table 2 show that AE2 protein abundance in the renal cortex was markedly decreased in response to chronic KHCO3 loading (49 ± 8% in KHCO3-loaded rats versus 100 ± 7% in control group; P < 0,01). No significant change was observed in the abundance of AE2 in the outer medulla after KHCO3 loading (130 ± 19% in KHCO3-loaded rats versus 100 ± 10% in control group).
Table 3. Functional data in rats after chronic administration of 0.28 M KCl or KHCO3a
Figure 5. Effects of chronic KHCO3 loading on AE2 protein abundance in the rat kidney. (A) Immunoblots of membrane fractions from the cortex obtained from six KHCO3-loaded rats (k) and their respective controls (c). Each lane was loaded with 10 μg of protein. Immunoblots were reacted with anti-AE2 antibody and revealed the expected 180-kD band. (B) Densitometric analyses from six independent experiments showing that the abundance of AE2 in KHCO3-loaded rats was significantly decreased in the cortex but not in the outer medulla when compared with control rats. Bars represent means ± SEM. *Statistically significant increase versus control (P < 0.05).
Rats with Chronic KCl Loading Did not Experience Changes in the Abundance of AE2
Our findings in the renal cortex could also be interpreted to indicate that Cl− loading upregulates AE2 independent of acid-base or Na+ balance. Indeed, as summarized in Table 2, we observed upregulation of AE2 expression in response to NH4Cl and NaCl loading, whereas there was no effect on AE2 expression when sodium loading was achieved with NaHCO3. To test the hypothesis that AE2 expression is regulated by chloride balance, we next studied the effects of an equivalent chloride load administered with K+ as the accompanying cation rather than Na+. As expected, high dietary KCl intake markedly increased urinary chloride excretion from the control value of 10 ± 2 to 60 ± 9 mmol/L (P < 0.05; Table 3), an increase similar in magnitude to those observed during loading with NaCl and with NH4Cl (see Table 1). Importantly, the potassium load reflected by urinary potassium excretion was identical to that achieved by KHCO3 loading, but the KCl treatment did not alter acid-base status (Table 3). As shown in Figure 6, KCl loading had no significant effect on AE2 abundance in either the renal cortex (129 ± 10% in KCl-loaded rats versus 100 ± 9% in control group; NS) or the outer medulla (143 ± 18% in KCl-loaded rats versus 100 ± 15% in control group; NS). The data thus indicate that neither Cl− nor K+ loads are important regulators of AE2 abundance.
Figure 6. Effects of chronic KCl loading on AE2 protein abundance in the rat kidney. (A) Immunoblots of membrane fractions from the cortex obtained from six KCl-loaded rats (kcl) and their respective controls (c). Each lane was loaded with 10 μg of protein. Immunoblots were reacted with anti-AE2 antibody and revealed the expected 180-kD band. (B) Densitometric analyses from six independent experiments showing that the abundance of AE2 in KCl-loaded rats is not modified in the cortex or in the outer medulla as compared with control rats. Bars represent means ± SEM.
Taken together, our results show that AE2 protein expression is appropriately regulated by acid-base status in the renal cortex, i.e., stimulated by metabolic acidosis and inhibited by alkalemia. In the outer medulla, the effect of acid-base balance on AE2 abundance is limited to the stimulatory effect of acidosis. Nevertheless, our experiments also demonstrate that acid-base balance is not the only determinant of AE2 regulation. Indeed, Na+ intake seems to exert a potent effect also on AE2.
Immunohistochemistry
AE2 is mainly expressed on the basolateral membranes of the TAL, macula densa, and inner medullary collecting duct. To determine at which of these sites NH4Cl and NaCl loading increased AE2 expression, we performed immunohistochemistry in control and treated rats. As previously shown (11,15⇓), AE2 immunolabeling with the AE2a/b antibody in kidneys from control rats was evident in basolateral domains of both cortical and medullary segments of TAL, as well as in the macula densa, and also in the terminal collecting ducts. After the different treatments, all of the detectable changes in the intensity of AE2 labeling were observed in the TAL; we also did not observe the apparition of an AE2 labeling in cells that did not express AE2 in the basal state. Kidneys from NaCl-loaded rats showed markedly increased intensity of AE2 labeling in basolateral domains of MTAL, with a lesser increase noted in cortical TAL (CTAL; Figure 7). In sections from NH4Cl-loaded rats, a marked increase in labeling intensity was evident in the CTAL, and the labeling in CTAL extended to more distal parts of the CTAL (Figure 8B) as compared with controls (Figure 8A). No changes in AE2 labeling were observed in MTAL in NH4Cl-loaded rats (Figure 8D) compared with controls (Figure 8C).
Figure 7. AE2 immunolabeling in kidneys from control (A and C) and NaCl-loaded (B and D) rats. In kidneys from NaCl-loaded rats, the intensity of AE2 labeling in basolateral domains of cortical thick ascending limb (CTAL; A and B) was weakly increased, whereas a marked increase in labeling intensity was evident in medullary thick ascending limb (MTAL; C and D). CD, collecting duct. Scale bars = 50 μm.
Figure 8. AE2 immunolabeling in kidneys from control rats (A and C) and NH4Cl-loaded rats (B and D) rats. In cortex, AE2 labeling was increased in the CTAL in NH4Cl-loaded rats (B) in comparison with controls (A). In MTAL, no changes were seen in the intensity of AE2 labeling in NH4Cl-loaded rats (D) when compared with controls (C). Scale bars = 50 μm.
Discussion
The AE2 Cl−/HCO3− exchanger polypeptides are expressed throughout the distal nephron in epithelial cell basolateral membranes, with highest expression in the MTAL, macula densa, and inner medullary collecting duct (11,15⇓). Lower levels of AE2 expression were detected in CTAL, thin limbs, and principal cells of the outer medullary collecting duct (11). AE2 may contribute to transepithelial bicarbonate transport, to chloride secretion, or to “housekeeping functions” such as cellular regulatory volume increase and pHi regulation. In two studies using basolateral membrane vesicles isolated from a purified MTAL tubule suspension, we previously demonstrated that the main basolateral HCO3− efflux pathway in MTAL cells is via Cl−/HCO3− exchange activity attributable to AE2 (12,13⇓). An additional study from our laboratory confirmed in isolated, intact, microperfused MTAL that the main basolateral bicarbonate efflux pathway was Cl− dependent, Na+ independent, and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) sensitive, properties shared with AE2 (16).
The present study examined the regulation of AE2 expression in rats that were subjected to chronic acid-base perturbations and increased sodium intake. Chronic metabolic acidosis markedly increased AE2 abundance in the renal cortex and outer medulla, as detected by immunoblot. Chronic sodium loading, as either NaHCO3 or NaCl, increased AE2 abundance to a similar extent in the outer medulla, whereas in the cortex, only chronic NaCl loading led to an increase in AE2 protein expression. Immunohistochemistry was performed to define the nephron segments in which AE2 abundance was changed. First, the immunoblot data can be ascribed to changes in the basolateral membrane of the TAL because AE2 was not found to be upregulated in other segments. Second, immunoblot and immunohistochemistry findings were in agreement, with the exception of those of the outer medulla after NH4Cl loading. Discrepancy between immunoblotting and immunohistochemistry regarding AE2 regulation in the inner stripe of the outer medulla could be explained by the lower sensitivity of immunohistochemistry than of immunoblotting for quantifying differences in protein abundance. However, although AE2 immunostaining intensity in TAL of the inner stripe of the outer medulla was not increased in NH4Cl-loaded rats, the tubules seemed larger than in control rats (i.e., hypertrophic). This latter observation could explain detection of increased AE2 abundance only by immunoblot. In contrast, in NaCl-loaded rats, TAL show a marked increase in immunostaining intensity, confirming that AE2 protein abundance was specifically upregulated in this condition in the outer medulla. Finally, the immunoblot data suggest considerable variation among individuals in the magnitude of AE2 upregulation in the outer medulla (see Figure 2). This could explain why individual fixed kidneys studied by immunohistochemistry can show little or no regulation.
In a recent study, Frische et al. (15) determined the renal expression pattern of polypeptide products of the AE2 transcript variants AE2a and AE2b and the regulation of AE2 polypeptides by chronic acid loading. In that study, levels of AE2 polypeptide were markedly increased in CTAL in response to chronic administration of NH4Cl or HCl. In contrast to our current results, Frische et al. observed no change in AE2 protein level in the outer medulla. However, whereas the 6-d acid-loaded rats in the series studied by Frische et al. exhibited only a decrease in urinary pH without altered plasma acid-base status [see the Methods section in reference (15)], our “acidotic” rats exhibited a frank metabolic acidosis, with significant decreases in both plasma pH and bicarbonate concentration (see Table 1). The greater degree of acidosis experienced by the animals in our current study may account for the greater increase in AE2 expression [in the renal cortex: 644 versus 203% in ref (15)], including upregulation of AE2 not only in CTAL but also in MTAL. Another possible explanation for the detection of increased AE2 in MTAL in our current study may lie in our use of immunoblot experiments of a particular fraction more highly enriched in plasma membrane. Indeed, comparison of whole homogenates from the inner stripe of outer medulla of our control and NH4Cl-loaded rats revealed only minimal increase in AE2 abundance (132 ± 7% in the acidotic group versus 100 ± 14% in the control group; NS), similar to the findings of Frische et al. (15).
The upregulation of AE2 seen in the MTAL in response to loading with NH4Cl and to Na+ loading is a novel finding. Good (7) previously demonstrated in isolated MTAL microperfused in vitro that bicarbonate absorption is increased by chronic NH4Cl loading. He also observed that chronic sodium intake increased bicarbonate absorptive capacity to the same extent when sodium was administered as either sodium bicarbonate or sodium chloride. Thus, the increase in AE2 abundance demonstrated here is likely to have contributed to the previously observed increase in HCO3− absorption. Upregulation of basolateral bicarbonate efflux through AE2-mediated Cl−/HCO3− exchange may, with coordinated stimulation of apical NHE3-mediated Na+/H+ exchange, contribute to enhanced transepithelial bicarbonate reabsorption during metabolic acidosis (10). In contrast, the stimulatory effect of chronic sodium loading on bicarbonate reabsorption could not be due to the same mechanism because NHE3 abundance in TAL is decreased or unaltered in the TAL during chronic NaHCO3 or NaCl loading (10,17⇓). During chronic changes in sodium bicarbonate loading, the increases in bicarbonate absorption and in AE2 abundance seem inappropriate for maintaining acid-base balance and likely reflect MTAL adaptation to high sodium intake. When chloride or HCO3− loads were administered as K+ salts, no change in AE2 abundance was seen in the outer medulla, demonstrating the importance of increased sodium intake in AE2 adaptation. These results also show that the high chloride load that accompanies NH4+ has no effect on AE2 expression and so demonstrate that acidosis per se regulates AE2 expression. Although chronic KHCO3 loading and its mild metabolic alkalosis did not lead to AE2 downregulation in the outer medulla, severe alkalemia was shown to decrease bicarbonate absorptive capacity in the MTAL (9). It thus seems that acid-base status is another important determinant for AE2 adaptation.
In the present study, NaHCO3 loading was associated with a suggestive but nonsignificant trend toward hypokalemia (Table 1), reflecting a possible potassium depletion. Hypokalemia is known to stimulate HCO3− reabsorption (18,19⇓). NHE3, which plays an important role in the process of HCO3− reabsorption, is upregulated after 4 d of dietary K+ depletion–induced hypokalemia (20). Thus, upregulation of AE2 abundance in NaHCO3-loaded rats may also be caused by or reflect an effect of K+ depletion and its associated increased HCO3− absorption. Taken together, these results support the hypothesis that HCO3 transport by MTAL cells through AE2 regulation is specifically regulated by changes in sodium intake and acid-base status and not by changes in chloride and potassium intake.
The CTAL response to salt loading differed from that of the MTAL. NaHCO3 loading produced no change in CTAL AE2 abundance. In contrast, the effect of NH4Cl loading was much greater in CTAL than in MTAL. The unchanged renal cortical AE2 abundance after NaHCO3 loading likely reflects the sum of two processes: AE2 downregulation by alkali load and AE2 upregulation by the concomitant Na load. This possibility is further supported by the decrease in renal cortical AE2 abundance in response to KHCO3 loading and by the absence of effect of KCl loading. Thus, AE2 expression in the renal cortex may be particularly sensitive to changes in acid-base status.
Because metabolic acidosis decreases filtered bicarbonate load while increasing proximal bicarbonate reabsorptive capacity, upregulation of TAL bicarbonate transport in metabolic acidosis might seem unnecessary, but the increase in bicarbonate transport capacity during acidosis could enhance urinary acid excretion by maintaining a low luminal bicarbonate concentration along the TAL and further limit bicarbonate delivery to the distal tubule. In particular, the adaptive increase in bicarbonate absorption (e.g., AE2 and NHE3 proteins adaptation) may be coupled to ammonium handling. As previously proposed by Good (7), TAL bicarbonate absorptive capacity may compensate for the acidosis-induced increase in ammonium absorption along the TAL that would tend to alkalinize the luminal fluid were not H+ secretion simultaneously increased.
The present study provides additional support for the importance of cortical distal nephron segments in the renal regulation of acid-base status. Previous studies have shown that chronic metabolic acidosis markedly increases ammonia secretion along the collecting duct (21–24⇓⇓⇓), but a microcatheterization study suggested that in acidotic rats, the bulk of ammonia reenters the luminal fluid primarily in the cortical collecting duct (22). In that study, the amount of ammonium secreted into the cortical collecting duct of acidotic rats was 15-fold greater than that of control rats, whereas the ammonium added to the medullary collecting duct was increased only three- to fourfold. Thus, if HCO3− reabsorption by the TAL is regulated to support parallel increase in ammonium transport, then a correspondingly marked increase in HCO3− transport capacity should occur in the cortical portion of the TAL.
The dependence of both TAL luminal acidification and AE2 upregulation on sodium intake suggests that the TAL segment may play an important role in maintaining acid-base balance when sodium chloride intake is altered. In the setting of elevated salt intake and extracellular fluid volume, an increase in bicarbonate absorption in TAL could oppose a reduction in collecting duct proton secretion as a result of decreased plasma aldosterone levels to maintain urinary acid excretion. The mediators of the TAL adaptation in response to changes in sodium intake have not been determined in this study.
In summary, our results demonstrate that acid-base balance and sodium intake are important and independent determinants of AE2 regulation in the TAL. This study also supports the importance of the CTAL in the renal regulation of acid-base status. The CTAL and MTAL may play an especially important role in maintaining acid-base balance when sodium intake is altered. We propose that this process involves changes in the basolateral expression of the Cl−/HCO3− exchanger AE.
Acknowledgments
The Water and Salt Research Center at the University of Aarhus is established and supported by the Danish National Research Foundation (Danmarks Grundforskningsfond). Additional funding was provided by the European Commission (QLRT-2000-00987).
We thank Susan M. Wall for providing the AE2a/b antibody and Inger Merete Paulsen, Ida Maria Jalk, and Lotte Vallentin Holbech for expert technical assistance.
- © 2004 American Society of Nephrology
References
Jump to section
More in this TOC Section
Cited By...
- Regulation of WNK1 Expression by miR-192 and Aldosterone
- Exposure to Maternal Diabetes Induces Salt-Sensitive Hypertension and Impairs Renal Function in Adult Rat Offspring
- Inhibition of Estrogen-Mediated Uterine Gene Expression Responses by Dioxin
- Pendrin Regulation in Mouse Kidney Primarily Is Chloride-Dependent