Metabolic acidosis is associated with alteration in fluid and electrolyte reabsorption in a number of nephron segments. However, the effects of metabolic acidosis on urine osmolality and aquaporin-2 (AQP-2) remain poorly understood. In these studies, we examined the effects of chronic metabolic acidosis on water handling by the kidney. Rats were placed in metabolic cages and subjected to water (control) or 280 mM NH4Cl loading for 120 h to induce metabolic acidosis. The results indicated a significant increase in urine osmolality with no change in urine volume or urinary Na+ excretion in acid-loaded animals. This effect was independent of alteration in fluid intake or salt/Cl- loading. Immunoblotting and Northern hybridization studies indicated that AQP-2 protein abundance and mRNA expression levels increased significantly along the collecting duct system of NH4Cl-but not NaCl-loaded animals. RIA results indicated that metabolic acidosis was associated with a fourfold increase in circulating levels of vasopressin (AVP) and a significant increase in brain AVP mRNA expression levels. In conclusion, metabolic acidosis upregulates the expression levels of AQP-2 and increases urine osmolality, suggesting an adaptive increase in water reabsorption in the collecting duct. A concomitant increase in AVP synthesis and secretion likely plays an essential role in the adaptation of AQP-2 in metabolic acidosis.
- urine osmolality
- sodium excretion rate
several studies have examined the effect of metabolic acidosis on fluid and electrolyte reabsorption in various nephron segments. Chronic metabolic acidosis increased reabsorption in the proximal tubule (1, 19, 27), medullary thick ascending limb (mTAL) (20), and collecting duct (10, 46). In proximal and mTAL tubules, the increase in reabsorption in acidosis was predominantly mediated via upregulation of the apical Na+/H+ exchanger (NHE3) (2, 29, 53). Interestingly and despite an increase in reabsorption, metabolic acidosis was consistently shown to decrease water and NaCl reabsorption in the proximal tubule (30, 43, 51). The decrease in Na+ reabsorption was attributed to the downregulation of organic anion pathways that are coupled to apical NHE3 (51), Na+-dependent inorganic sulfate (Na-Si) cotransporter (40), and Na+-phosphate cotransporter (NaPi-2) (7). Despite the known effects on ion transporters in the proximal tubule and mTAL, little is known about the effect of metabolic acidosis on water transport in the collecting duct. Recent studies examined the effects of metabolic acidosis on V1a and V2 vasopressin (AVP) receptors in the kidney and reported an increase in V1a and a downregulation of V2 expression in response to metabolic acidosis (48). These studies, however, did not examine the effects of metabolic acidosis on water metabolism, aquaporin-2 (AQP-2) expression, or AVP synthesis and secretion.
In addition to acid-base regulation, the kidney plays an important role in the maintenance of water homeostasis by increasing water excretion in states of excessive water intake and water conservation in states of dehydration. The regulation and maintenance of water balance by the kidney depend on the activity of water transport proteins called aquaporins (AQPs). At least seven isoforms of AQPs are expressed in the kidney: AQP-1, AQP-2, AQP-3, AQP-4, AQP-6, AQP-7, and AQP-8. AQP-1 is constitutively expressed in apical and basolateral membranes of the proximal tubule and descending limb cells as well as in endothelial cells of descending vasa recta (36). AQP-2 is the AVP-regulated water channel predominantly expressed in the apical surface of principal cells, with strong labeling also in the intracellular and subapical vesicle in the connecting tubule and the entire collecting duct system (34). This distribution is consistent with the rapid regulation of water reabsorption through apical AQP-2 by AVP (33, 34). AQP-3 and AQP-4 are expressed in the basolateral membrane and mediate the exit of water from collecting duct principal cells (16, 24). AQP-6 is present in the collecting duct intercalated cells (55), AQP-7 is abundant in the brush border of the proximal tubule (23), and AQP-8 is expressed at a low level in proximal tubule and collecting duct principal cells (17). AQP-7 is likely involved in isosmotic water reabsorption in the proximal tubule, whereas the physiological role of AQP-6 and AQP-8 remains unclear (35). Studies in rats demonstrated that AQP-6 expression is regulated in response to altered acid-base balance or water intake and suggested that AQP-6 may play a role in the maintenance of acid-base and water metabolism (39).
Extensive studies demonstrated that, among these AQPs, AQP-2 is the most regulated protein in response to various physiological or pathophysiological conditions associated with altered water balance and/or urine osmolality (see Ref. 35 for review). In addition, mutation or deletion of the gene encoding AQP-2 protein demonstrated its crucial role in the urinary concentrating mechanism. A point mutation in AQP-2 (T126M) was identified as a cause of non-X-linked recessive nephrogenic diabetes in humans (32). Recent studies demonstrated that mice bearing the AQP-2 T126M mutation develop a significant urinary concentrating defect that is resistant to AVP (54). The downregulation of AQP-2 is also involved in the central and nephrogenic diabetes insipidus developed as a result of mutations in AVP and its receptor V2 genes, respectively (11, 14).
In the present study, the effects of metabolic acidosis on water balance, urine osmolality, and urinary Na+ excretion were determined. Metabolic acidosis was induced using the established protocol of NH4Cl loading, and the specificity of its effects on the above-mentioned physiological parameters was determined using pair-watering and pair-salt-loading protocols. In additional experiments, the expression pattern of apical water channels (AQP-1 and AQP-2) was determined, and the brain mRNA and circulating levels of AVP were measured.
Chronic Metabolic Acidosis
The experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Cincinnati.
Male Sprague-Dawley rats (180–210 g) were placed in metabolic cages and allowed free access to regular rat chow and distilled water. After 72 h of adjustment in metabolic cages, rats were divided into two groups: one group (control) remained on distilled water, and the second group was switched to a drinking solution containing 280 mM NH4Cl to induce metabolic acidosis as previously described (2, 3, 7, 8, 20, 29, 51). Rats were given food and drinking fluid once daily (at 5 PM), and urine was collected under mineral oil. The animals were maintained in a temperature-controlled room regulated on a 12:12-h light-dark cycle for the duration of the experiment. Water intake, urine volume, urine osmolality, urine Na+ concentration, and urine pH were measured daily. After 120 h of treatment, rats were killed, and blood was obtained from the heart for blood composition analysis. The kidneys were removed, and the superficial cortex, inner stripe of the outer medulla, and inner medulla were dissected and snap frozen in liquid nitrogen and stored at -80°C for total cellular RNA and protein isolation.
A second set of experiments was performed as described above, except the animals were subjected to water and salt restriction. Accordingly, male Sprague-Dawley rats were placed in metabolic cages with free access to food and water and randomly divided into three groups: the first group had free access to NH4Cl solution, the second group was water restricted, and the third group was given restricted access to a solution containing 280 mM NaCl. All rats in these groups had free access to food, and the experiment was terminated 120 h after the beginning of the treatment. The rationale for this experiment is to provide pair-watering and pair-salt-loading controls for the NH4Cl-loaded animals (see results). During this experiment, and in addition to the above-mentioned physiological parameters, body weight and food intake were measured daily.
Blood Composition and Urine Analyses
Serum electrolyte, creatinine, and blood urea nitrogen levels were measured using commercial services (Health Alliance Laboratory Services, Cincinnati, OH). Plasma and urine osmolality were measured by freezing point-based osmometry with a microosmometer (Advanced Instruments, Norwood, MA). Urine Na+ and K+ concentrations were measured with an automatic flame photometer (model IL943, Instrumentation Laboratory, Lexington, MA). Urine pH was measured with a pH meter (Accumet Basic AB15, Fisher Scientific, Hanover Park, IL).
Measurement of Plasma Levels of AVP
To measure plasma concentrations of AVP, rats were subjected to NH4Cl or normal water loading (6 animals in each group) and decapitated after 120 h. Blood was collected for plasma isolation, and the whole brain was dissected and rapidly frozen in liquid nitrogen and stored at -80°C for total RNA isolation. Plasma AVP concentration was determined by radioimmunoassay after a peptide extraction procedure according to the manufacturer's protocol (Peninsula Laboratories, San Carlos, CA).
Total RNA Isolation
Total cellular RNA was extracted from the renal cortex, the inner stripe of the outer medulla, and the inner medulla by the method of Chomczynski and Sacchi (12). Briefly, 0.5–1 g of tissue was homogenized at room temperature in 5–10 ml of TriReagent (Molecular Research Center, Cincinnati, OH). Total RNA was extracted by phenol-chloroform and precipitated by isopropanol. RNA was then quantitated by spectrophotometry and stored at -80°C.
Total RNA samples (30 μg/lane) were fractionated on a 1.2% agarose-formaldehyde gel and transferred to Magna NT nylon membranes using 10× sodium chloride-sodium phosphate-EDTA as transfer buffer. Membranes were cross-linked by ultraviolet light and baked for 1 h. Hybridization was performed according to Church and Gilbert (13) and as previously described (4–6). A PCR product fragment was used as specific probe for AQP-2 (nucleotides 102–397) as previously described (4). Coding sequence in AVP and renin genes (GenBank accession nos. M25646 and J02941, respectively) were used to design primers utilized to generate AVP- and renin-specific probes by RT-PCR using total RNA isolated from rat brain and kidney, respectively. For AVP, the primers were 5′-CCTCACCTCTGCCTGCTACTT-3′ (forward, nucleotides 77–97) and 5′-GGGGGGCGATGGCTCAGTAGAC-3′ (reverse, nucleotides 540–519). For renin, the primers were 5′-CTGCCACCTTGTTGTGTGAG-3′ (forward, nucleotides 1033–1052) and 5′-CCAGTATGCACAGGTCATCG-3′ (reverse, nucleotides 1296–1277). The PCR yielded a single band with expected sizes of 465 and 264 bp, corresponding to rat AVP and renin fragments, respectively, both of which were verified by sequencing (DNAcore, University of Cincinnati).
Preparation of Membrane Fractions From Renal Cortex, Outer Medulla, and Inner Medulla
A total cellular reaction containing plasma membrane and intracellular membrane vesicles was prepared from the cortex, inner stripe of the outer medulla, and inner medulla as previously described (3–5). The total protein concentration was measured, and the membrane fractions were solubilized at 65°C for 20 min and stored at -20°C.
Electrophoresis and Immunoblotting
Semiquantitative immunoblotting experiments were carried out as previously described (3–5). Briefly, the solubilized membrane proteins were size fractionated on 10% polyacrylamide minigels (Novex, San Diego, CA) under denaturing conditions. With the use of a transfer apparatus (Bio-Rad Laboratories, Hercules, CA), the proteins were electrophoretically transferred to a nitrocellulose membrane. The membrane was blocked with 5% milk proteins and then probed with affinity-purified anti-AQP-1 or anti-AQP-2. The secondary antibody was a donkey anti-rabbit IgG conjugated to horseradish peroxidase (Pierce). The site of antigen-antibody complexation on the nitrocellulose membranes was visualized using the chemiluminescence method (SuperSignal Substrate, Pierce) and captured on light-sensitive imaging film (Kodak). Bands corresponding to AQP-1 and AQP-2 proteins were quantitated by densitometric analysis using UN-SCAN-IT gel software (Silk Scientific, Orem, UT) and expressed as percentage of control. The densitometry values reported in results correspond to the level of expression of the nonglycosylated form of AQP-2 (29 kDa) water channel. The expression levels of native and glycosylated forms for AQP-2 and AQP-1 are illustrated in Figs. 5, 6, 7, 8, 9, 11, and 12. The equity in protein loading in all blots was first verified by gel staining using Coomassie brilliant blue R-250 (Bio-Rad).
Immunofluorescence Labeling of AQP-2
Immunofluorescence labeling with AQP-2 antibodies was performed according to established methods and as employed in our laboratory (38). Briefly, kidney sections from control or acid-loaded animals were obtained as described elsewhere (38). Primary AQP-2 antibody was diluted 1:60 in 1% BSA-0.3% Triton X-100-PBS solution and applied to sections overnight at room temperature. Sections treated with the primary antibody were rinsed and then incubated with a secondary antibody for 2 h at room temperature. Green fluorescent secondary Oregon green-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR) was used at a dilution of 1:150. Sections were then washed four times, air dried, and mounted in Vectashield mounting medium for fluorescence (Vector Laboratories, Burlingame, CA). Sections were examined, and images were acquired on the Nikon PCM 2000 laser confocal scanning microscope as 0.5- to 1-μm “optical sections” of the stained cell membrane. A ×20 objective was used. The standard argon laser 488-nm line and the 515/530-nm emission filters were used for the green-emitting dye. All sections from control and acidotic animals were processed on the same day and with the same dilutions of primary and secondary antibodies. More than 20 sections from 3 separate animals were examined in each group of control or acidotic animals.
[32P]dCTP was purchased from New England Nuclear (Boston, MA), nitrocellulose filters and other chemicals from Sigma Chemical (St. Louis, MO), and RadPrime DNA labeling kit from GIBCO BRL.
Semiquantification of immunoblot and Northern hybridization band densities was determined by densitometry using a scanner (ScanJet ADF, Hewlett Packard) and UN-SCAN-IT gel (Silk Scientific) and ImageQuaNT (Molecular Dynamics, Sunnyvale, CA) software, respectively. Data are expressed as percentage of control. Values are means ± SE. Statistical significance between experimental groups was determined by Student's t-test or one-way ANOVA as needed. P < 0.05 was considered significant.
The drinking solution containing 280 mM NH4Cl is extensively used to induce metabolic acidosis (2, 3, 7, 8, 20, 29, 51). Rats receiving NH4Cl developed a significant hyperchloremia and metabolic acidosis compared with controls (Table 1). No significant differences were observed between control and acidotic rats with respect to serum Na+, K+, creatinine, Ca2+, and plasma osmolality. Water- and NaCl-restricted animals had normal acid-base status and normal serum electrolytes compared with controls. The presence of metabolic acidosis clearly suggests that the animals are reasonably loaded with NH4Cl (Table 1).
Water Balance and Urine Osmolality in Control and Acidotic Rats
The average baseline volume of water intake was 31 ± 0.6 ml/24 h in rats receiving normal water (Fig. 1A). After the animals were switched to NH4Cl, fluid intake initially decreased to 24 ± 1.1 ml/24 h (P < 0.05 compared with baseline) and then returned to normal level (33 ± 3.1 ml/24 h, P > 0.05) within 120 h (n = 9 rats; Fig. 1A). Urine volume did not change at any time after the animals were switched to NH4Cl (n = 9; Fig. 1B). However, urine osmolality increased from 1,269 ± 44 mosmol/kgH2O at baseline to 2,185 ± 77 mosmol/kgH2O after 48 h (n = 9, P < 0.001) and plateaued at 1,901 ± 72 mosmol/kgH2O after 120 h of NH4Cl loading (P < 0.01 compared with baseline, n = 9 rats; Fig. 1C). Urine pH significantly decreased after 24 h of NH4Cl loading (P < 0.05 vs. baseline) and further decreased after 48 h (P < 0.001 vs. baseline) before recovering slightly after 120 h of treatment (Fig. 1D). These parameters remained unchanged in control rats receiving distilled water for the duration of the experiment (data not shown).
Effects of the Initial Decrease in Fluid Intake or Salt/Cl- Loading on Urine Volume and Urine Osmolality
The increased urine osmolality in NH4Cl-loaded rats (Fig. 1C) can result from the initial drop in fluid intake (Fig. 1A), Cl- loading, or increased osmotic diuresis resulting from decreased salt and fluid reabsorption in the proximal tubule in NH4Cl loading, or it could be specific to the presence of metabolic acidosis. To test these possibilities, rats were placed in metabolic cages with free access to food and water. At steady state, rats were divided into three groups: the first group remained on water (n = 5), the second group was switched to a drinking solution containing 280 mM NaCl (n = 5), and the third group had free access to 280 mM NH4Cl solution. All groups had free access to rat chow; however, access to water and NaCl was restricted to mimic the time course changes in fluid intake observed in NH4Cl-loaded animals (Fig. 1A).
After 120 h of treatment, the animals on water or NaCl restriction did not show any alteration in acid-base balance (Table 1), whereas NH4Cl-treated rats developed metabolic acidosis (Table 1). An initial water restriction (Fig. 2A) is associated with only a minor change in urine volume (Fig. 2B) and urine osmolality (Fig. 2C) within the first 48 h, which returned to baseline levels after 120 h. Interestingly, rats switched to NaCl solution showed a small but significant and sustained increase in urine volume (Fig. 3B), despite the initial 25% reduction in their fluid intake (Fig. 3A). Urine osmolality, however, remained unchanged for the duration of the experiment (Fig. 3C). Water balance and urine osmolality data for NH4Cl-loaded rats are included in Fig. 1. The volume of fluid intake in rats with free access to water or NH4Cl solution or in rats with restricted access to water or NaCl solution was not significantly different.
Rats with free access to a 280 mM NaCl solution showed a threefold increase in fluid intake and urine volume with a slight but significant reciprocal decrease in urine osmolality within 120 h of treatment (data not shown).
Food Intake, Body Weight, and Urinary Na+ Excretion Rate
Basal food intake was comparable between control and acidotic rats (∼15 g; Fig. 4A, Table 2). However, a slight reduction in food intake from day 1 to the end of the experiment was observed in NH4Cl-loaded animals (Fig. 4A, Table 2). Control rats maintained a normal growth rate for the duration of the experiment (Fig. 4B), whereas a slight decrease and delay in body weight gain were observed in rats switched to NH4Cl solution (Fig. 4B, Table 2). Food intake was higher in water-restricted rats than in all other groups; this correlates with a higher body weight gain in this group (Table 2). Rats subjected to NaCl loading showed a significant decrease in food intake (compared with other groups) and did not gain weight during 120 h of treatment (Table 2).
Interestingly, despite a significant inhibition of Na+ reabsorption in the proximal tubule in acidosis, urine Na+ excretion was comparable in acidotic (NH4Cl), control (water), and water-restricted animals (Table 2). However, and as expected, NaCl-treated animals showed a nearly fourfold increase in Na+ excretion (compared with other groups; Table 2), which likely results from increased osmotic diuresis induced by high NaCl intake (Table 2).
Molecular Regulation of Apical Water Channels (AQP-2 and AQP-1) in Metabolic Acidosis
AQP-2 expression in the cortex. Figure 5 shows immunoblot and Northern hybridization of AQP-2 in the cortex of kidneys harvested from control and acidotic animals. The abundance of AQP-2 protein increased ∼2.7-fold in acidotic vs. control rats (100 ± 27 vs. 368 ± 21%, P < 0.001, n = 4 for each; Fig. 5A). A Northern hybridization experiment (Fig. 5B) indicates that the mRNA expression of AQP-2 also increased significantly in acidotic rats (P < 0.02 vs. control, n = 4 for each).
AQP-2 expression in the outer medulla. Figure 6 shows immunoblot and Northern hybridization of AQP-2 in outer medulla of control and acidotic animals. The results indicate that acidosis increased AQP-2 protein abundance significantly compared with control (100 ± 11 vs. 188 ± 7%, P < 0.02, n = 4 for each; Fig. 6A). This Northern hybridization experiment indicates a significant increase in the mRNA expression levels of AQP-2 in acidosis (P < 0.001 vs. control, n = 4 rats for each; Fig. 6B).
AQP-2 expression in the inner medulla. Figure 7 shows immunoblot and Northern hybridization of AQP-2 in the kidney inner medulla of control and acidotic animals. AQP-2 protein abundance increased nearly twofold in acidotic rats (from 100 ± 36 to 282 ± 43% from control to acidosis, n = 4 for each, P < 0.02; Fig. 7A). The increase in AQP-2 protein correlated with the increase in its mRNA expression: AQP-2 mRNA increased by ∼1.5-fold in acidotic vs. control animals (n = 2 for each group; Fig. 7B).
AQP-1 expression in the cortex, outer medulla, and inner medulla. Membrane fractions that were utilized for AQP-2 expression were used for AQP-1 analysis. The results indicate that AQP-1 protein abundance remained unchanged in the cortex (Fig. 8A), outer medulla (Fig. 8B), and inner medulla (Fig. 8C) of acidotic vs. control rats.
Expression of AQP-2 in Whole Kidneys of Rats Subjected to NH4Cl Loading or Restricted Access to Water or NaCl Solution
Whole kidneys were removed from the animals subjected to water restriction, NaCl (280 mM) restriction, or NH4Cl (280 mM) loading as described above and used for total cellular protein and total mRNA isolation. Immunoblotting and Northern hybridization experiments were then performed to examine the protein abundance and mRNA expression levels, respectively, of AQP-2 in these groups. The results depicted in Fig. 9 indicate that AQP-2 protein abundance (Fig. 9A; P < 0.001) and mRNA expression levels (Fig. 9B; P < 0.001) were significantly increased in NH4Cl-but not NaCl-loaded rats compared with water-restricted rats. AQP-2 protein abundance was not altered in water-restricted vs. control (those allowed access to water) rats (P > 0.05; Fig. 9C).
Localization of AQP-2 in the Apical Membrane of Principal Cells in Acid-Loaded Rats
The activity of AQP-2 is mainly dependent on its localization in the apical plasma membrane of principal cells. Immunofluorescence labeling was carried out to examine the cellular distribution of AQP-2 protein in response to metabolic acidosis. The results shown in Fig. 10 indicate that AQP-2 protein is present in the apical membrane of principal cells and that the labeling is sharper and more abundant in acidotic than in control animals. Furthermore, there is no enhancement of intracellular AQP-2 staining in kidneys of acidotic animals. These results strongly suggest that increased AQP-2 expression in acidosis predominantly reflects enhanced membrane-bound AQP-2 abundance.
Effects of Metabolic Acidosis on Circulating Levels and mRNA Expression of AVP
The above results demonstrate that metabolic acidosis is associated with an upregulation of AQP-2 at the protein and mRNA levels. Previous studies have shown that collecting duct principal cells express AVP V2 receptor and that AQP-2 is regulated by AVP (14, 34). Hence, in the next set of experiments, the plasma concentration and the brain mRNA expression levels of the preprohormone of AVP were determined by radioimmunoassay and Northern hybridization studies, respectively. The results depicted in Fig. 11 indicate that chronic metabolic acidosis was associated with a fourfold increase in the plasma concentration of AVP in acidotic (n = 6) vs. control (n = 6) rats (P < 0.02; Fig. 11A). The increase in circulating AVP levels correlates with a significant increase in mRNA expression of the preprohormone of AVP in acid-loaded (n = 5) vs. control (n = 4) animals (P < 0.01; Fig. 11B). The mRNA expression of AVP did not change in the brain of animals subjected to water or salt restriction compared with the brain of control rats with free access to water (P > 0.05, data not shown).
Mechanism(s) by Which Metabolic Acidosis Increased Plasma AVP Concentration
As described above, plasma osmolality did not change significantly in acidotic rats, which suggests that the increase in the circulating levels of AVP is likely mediated via a nonosmotic stimulus. To determine whether increased AVP levels in metabolic acidosis were due to volume depletion, the expression levels of kidney renin mRNA were examined as an index of extracellular volume status (45, 47). The results depicted in Fig. 12 indicate that kidney renin mRNA levels were not altered in acidotic vs. control animals (P > 0.05, n = 4 for each). The kidney renin mRNA levels, however, were significantly increased in Na+-deprived animals (244 ± 8.9 vs. 100 ± 4.5%, P < 0.0002, n = 5 rats for each; Fig. 12) and in water-deprived rats (216 ± 8 vs. 100 ± 15%, P < 0.005, n = 5 for each group; Fig. 12). Na+ depletion and water deprivation are the two well-known conditions to be associated with extracellular volume depletion. These findings suggest that the increase in the circulating levels of AVP in metabolic acidosis is likely mediated via a mechanism that is independent of osmotic stress or extracellular volume status.
The results reported in this study demonstrate that chronic NH4Cl loading-induced metabolic acidosis in the rat is associated with a significant increase in urine osmolality (Fig. 1C). The increase in urine osmolality in acid-loaded rats correlates with a decrease in urine pH (Fig. 1D) and is independent of changes in fluid intake (Fig. 2). Rats subjected to a pair-NaCl loading, which exhibited a significant osmotic diuresis as indicated by polyuria (Fig. 3B) and natriuresis (Table 2) without metabolic acidosis (Table 1), did not show an increase in urine osmolality (Fig. 3C). This indicates that the increase in urine osmolality in metabolic acidosis is not secondary to the increase in the fluid load delivered to the distal nephron as a result of decreased fluid and salt reabsorption in the proximal nephron (Osmotic diuresis). Hence, it is highly possible that the increased urine osmolality is secondary to the metabolic acidosis that develops as a result of NH4Cl loading (Table 1).
Several recent studies examined the effect of NH4Cl loading on urine osmolality and showed conflicting results. In agreement with our results, rats subjected to NH4Cl loading (NH4Cl included in the diet) for 7 days showed a significant increase (∼2-fold) in urine osmolality and solute-free water reabsorption with no change in urine volume (25, 28, 39). While acid-base status of the animals was not determined in these studies, urine pH was significantly lower in NH4Cl-fed animals (28, 39). In rats loaded with 280 mM NH4Cl in the drinking water for 2 wk, polydipsia and polyuria with no change in urine osmolality were observed (28). The discrepancy between these findings and the present results is likely due to a difference in the duration of NH4Cl treatment (2 wk vs. 5 days). Another plausible, although less likely, explanation is that the development of polydipsia could alter AVP secretion and decrease AQP-2 expression, which in turn could lead to polyuria.
The enhanced urine osmolality in acid-loaded rats likely results from increased water reabsorption in the collecting duct, which is supported by an adaptive upregulation of AQP-2 expression in metabolic acidosis. Indeed, semiquantitative immunoblotting studies revealed that AQP-2 protein abundance was significantly increased in the cortex (Fig. 5A), outer medulla (Fig. 6A), and inner medulla (Fig. 7A) of acidotic animals. Northern hybridization experiments indicated that the increase in AQP-2 protein abundance correlated with a significant increase in the expression of AQP-2 mRNA in the collecting ducts of acidotic rats (Figs. 5B, 6B, and 7B). The increase in AQP-2 expression was observed in NH4Cl-but not in salt-loaded rats compared with pair-watered animals (Fig. 9), indicating that the upregulation of AQP-2 in these settings likely requires the presence of metabolic acidosis. On the basis of urine volume data, it appears that water reabsorption in the collecting duct is similar between control and acid-loaded rats. However, the load of fluid delivered to the distal nephron is much greater in acidotic than in control animals as a result of decreased fluid reabsorption in the proximal tubule by metabolic acidosis. Hence, the increase in AQP-2 expression in acidosis contributes to increased reabsorption of excess water that is delivered to the collecting duct and thus compensates for the defect in fluid transport in the proximal tubule. The upregulation of apical water channels by metabolic acidosis appears to be more specific for AQP-2, inasmuch as AQP-1 remained unchanged in all three regions of the kidney (Fig. 8).
The present studies also demonstrate for the first time that NH4Cl loading-induced metabolic acidosis in the rat is associated with an increase in the circulating levels of AVP (Fig. 11A) and that this correlates with a significant increase in the expression levels of AVP's preprohormone mRNA (Fig. 11B). Whether metabolic acidosis increases AVP synthesis via an increase in the transcription rate of its gene or through an increase in the stability of its mRNA remains unknown at this time.
The signaling pathway(s) mediating the rise in AVP synthesis and secretion in acidotic animals is yet to be determined. Blood urea nitrogen concentration was normal in acidotic animals (Table 1), strongly suggesting that volume depletion was not responsible for increased circulating AVP levels in acidotic animals. In support of this conclusion, we observed that kidney renin mRNA expression remained unchanged in acidosis (Fig. 12). Plasma osmolality was slightly but not significantly increased in acidotic vs. control animals (Table 1). It is possible that such a mild level of hypertonicity could trigger the stimulation of AVP synthesis and secretion. Alternatively, acute intravenous infusion of hydrochloric acid in fetal sheep (52) or dog (50) was associated with an increase in plasma AVP levels; however, no studies have examined the effect of chronic metabolic acidosis on serum antidiuretic hormone levels.
The increase in the circulating levels of AVP could play a major role in enhanced AQP-2 expression and water reabsorption in the collecting duct of acidotic animals. Indeed, in vivo and in vitro studies have demonstrated that AVP increases water reabsorption in the collecting duct predominantly, if not exclusively, through the apical water channel AQP-2. The regulation of AQP-2 by AVP involves two mechanisms: a short-term effect, which involves a rapid trafficking of AQP-2 from subapical vesicles to the apical plasma membrane of principal cells (33, 42), and a long-term effect, during which chronic administration of AVP increases the expression of AQP-2 and results in an increase in the number of AQP-2 water channels per collecting duct principal cell (14). This effect likely involves an increase in the transcription rate of the AQP-2 gene, which is stimulated by the cAMP-cAMP response element-binding protein pathway (22).
There is paucity of information with regard to the effects of metabolic acidosis on water metabolism and AVP secretion in humans. In this context, the results of the present work might have an important clinical significance. It is well established that metabolic acidosis impairs the reabsorption of water and NaCl in the proximal tubule. The upregulation of AQP-2 in metabolic acidosis reported here is likely a compensatory response to decreased fluid absorption in the proximal tubule and should increase water reabsorption in the collecting duct. This in turn minimizes water loss originating from impaired proximal tubule function. In addition to the regulation of AQP-2 and water transport, it is likely that Na+ reabsorption in the distal nephron is also stimulated in metabolic acidosis. This is suggested by a low urinary Na+ excretion rate (Table 2), despite the inhibition of NaCl reabsorption in the proximal tubule in acidosis. Recent in vivo and in vitro studies demonstrated that metabolic acidosis increased the expression and activity of mTAL apical Na+-K+-2Cl- cotransporter (9). In addition, several studies have shown that AVP treatment stimulates Na+ transport in the cortical collecting duct acutely (18, 21) and chronically via an increase in the protein abundance of the epithelial Na+ channel (ENaC) (15). Aldosterone, which is the main regulator of Na+ homeostasis by specifically targeting ENaC and thiazide-sensitive NaCl cotransporter (TSC or NCC) (26, 31), was also shown to increase in metabolic acidosis (37, 44). Moreover, aldosterone is well known to play an important role in water metabolism by promoting the hydrosmotic action of AVP in the collecting duct (41, 46). Hence, it is likely that these hormones stimulate Na+ and water reabsorption in the distal nephron and, thus, prevent Na+ and water wasting in metabolic acidosis.
In conclusion, NH4Cl-induced metabolic acidosis is associated with increased urine osmolality and enhanced expression of AQP-2 in the collecting duct. A concomitant increase in AVP synthesis and secretion in metabolic acidosis likely plays an essential role in this adaptive process. The increase in water and Na+ reabsorption in the distal nephron is a compensatory process and could prevent excessive fluid and Na+ loss that otherwise would result from decreased fluid reabsorption in the late proximal tubule in metabolic acidosis.
We thank Drs. L. Ma and S. Petrovic for help with the immunofluorescence labeling of AQP-2.
These studies were supported by the University of Cincinnati Academic Development Fund and by the Paul Teschan Fund, DCI, Inc. (to H. Amlal) and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-53548 (to S. Sheriff) and DK-54430 (to M. Soleimani).
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.
- Copyright © 2004 the American Physiological Society