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Physiol. Genomics 27: 271-281, 2006. First published July 25, 2006; doi:10.1152/physiolgenomics.00069.2006
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Received 24 April 2006; accepted in final form 27 June 2006.
Physiological Genomics 27:271-281 (2006)
1094-8341/06 $8.00 © 2006 American Physiological Society

Kidney collecting duct acid-base "regulon"

Lydie Cheval1, Luciana Morla1, Jean-Marc Elalouf2 and Alain Doucet1

1 Laboratoire de Physiologie et Génomique Rénales, Unité mixte de recherche 7134 Centre National de la Recherche Scientifique/Université Pierre et Marie Curie, Institut Fédératif de Recherche 58, Institut des Cordeliers, 15 rue de l'Ecole de Médecine, 75270 Paris cedex 6, France
2 Département de Biologie Joliot Curie, Commissariat à l'Énergie Atomique, 91191 Gif sur Yvette cedex, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Kidneys are essential for acid-base homeostasis, especially when organisms cope with changes in acid or base dietary intake. Because collecting ducts constitute the final site for regulating urine acid-base balance, we undertook to identify the gene network involved in acid-base transport and regulation in the mouse outer medullary collecting duct (OMCD). For this purpose, we combined kidney functional studies and quantitative analysis of gene expression in OMCDs, by transcriptome and candidate gene approaches, during metabolic acidosis. Furthermore, to better delineate the set of genes concerned with acid-base disturbance, the OMCD transcriptome of acidotic mice was compared with that of both normal mice and mice undergoing an adaptative response through potassium depletion. Metabolic acidosis, achieved through an NH4Cl-supplemented diet for 3 days, not only induced acid secretion but also stimulated the aldosterone and vasopressin systems and triggered cell proliferation. Accordingly, metabolic acidosis increased the expression of genes involved in acid-base transport, sodium transport, water transport, and cell proliferation. In particular, >25 transcripts encoding proteins involved in urine acidification (subunits of H-ATPase, kidney anion exchanger, chloride channel Clcka, carbonic anhydrase-2, aldolase) were co-regulated during acidosis. These transcripts, which cooperate to achieve a similar function and are co-regulated during acidosis, constitute a functional unit that we propose to call a "regulon".

serial analysis for gene expression; mouse; metabolic acidosis; V-ATPase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE RENAL CONTROL of acid-base balance integrates the glomerular filtration of acids and bases, the generation of ammonia along the proximal tubule, and complex processes of acid and/or base reabsorption and secretion along the nephron. Collecting ducts play a major role in acid-base homeostasis through the final control of bicarbonate, proton, and ammonium excretion (26, 56). For example, metabolic acidosis homeostasically enhances the reabsorption of bicarbonate and the secretion of proton and ammonium in the collecting duct (26, 50). The main objective of this work was to identify the molecular partners involved in acid-base transport in the collecting duct and its regulation. For this purpose, we searched for the genes that participate in the adaptation to metabolic acidosis using both transcriptome and candidate gene strategies.

Acid-base transport in the collecting duct originates from intercalated cells, whereas principal cells are responsible for sodium, potassium, and water transport (35). However, adaptation to metabolic acidosis alters transport processes in both cell types. Functional adaptations of the collecting duct also often include cell hypertrophy, hyperplasia, and transdifferentiation phenomena (1, 25, 30, 43). Therefore, to delineate the genes involved in the adaptation of acid-base transport from those involved in other processes, we compared the collecting duct transcriptomes of acidotic and potassium-depleted (LK) mice, since the latter display a hypertrophy/hyperplasia response, but no change in acid-base balance (9).

This study was restricted to the outer medullary collecting duct (OMCD), because it plays a major role in acid-base homeostasis and it contains a single type of intercalated cell (47). The OMCD transcriptome of acidotic mice was determined using the serial analysis for gene expression (SAGE) microassay previously developed in the laboratory (53) and compared with the OMCD transcriptomes of normal and LK mice previously deciphered with the same method (9).


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Animal experimentation was performed in accordance with French legislation. Experiments were carried out on male 8- to 10-wk-old C57BL/6J mice (Charles River Breeding Laboratories) fed ad libitum either a standard (A04, SAFE, Epinay, France) or an NH4Cl-supplemented diet (5 g powdered A04 diet, 0.2 g agarose, and 6 ml NH4Cl 0.7 M). Metabolic studies were performed in individual metabolic cages (Techniplast, Limonest, France) at 3 or 14 days after the onset of the treatment. After urine collection, blood was withdrawn by retro-ocular puncture. Urine and plasma biochemical parameters were determined with an automatic analyzer (Konelab 20i; Thermo, Cergy, France), except ammonium, determined by Berthelot's method, and blood acid-base parameters, measured with a blood-gas analyzer (AVL Compact 1; AVL Instruments Médicaux, Eragny/Oise, France). Urine aldosterone concentration was determined by radioimmunoassay. Glomerular filtration rate (GFR) was estimated by the clearance of creatinine.

Transcriptome analysis.
SAGE is a quantitative method of analysis of gene expression based on isolation and sequencing of short (10-bp) informative sequences specific for each cDNA (52). The validity of the method rests on the facts that a 10-bp nucleotide sequence tag is long enough to be specific for a transcript and that the relative abundance of transcript-specific tags in the library is representative of the cognate transcripts abundance in the biological material (52).

Approximately 650 OMCDs were microdissected, as previously described (9), from collagenase-treated kidneys of six mice fed the NH4Cl-supplemented diet for 3 days. The SAGE library was generated using the SAGE adaptation for downsized extracts, a microadaptation previously used for generating the control and LK libraries (9). Sequencing of DNA minipreps was performed by Genome Express (Meylan, France). Sequence files were analyzed with SAGE2000 software (52).

Tags were identified through matching to UniGene clusters using the SAGEmap resource (www.ncbi.nlm.nih.gov/SAGE) at the National Center for Biotechnology Information. The reliability of tag identification was validated by confirming the correct location and orientation of the tag in relation to the 3'-most Sau3AI site, as well as the presence of a polyadenylation signal and/or poly(A) tail.

mRNA extraction and real-time RT-PCR analysis.
RNAs were extracted, according to the technique previously described (15), from pools of 20–50 OMCDs microdissected from collagenase-treated kidneys of mice fed either the control diet or the NH4Cl-supplemented diet for 3 or 14 days. RNAs were reverse transcribed using the first-strand cDNA synthesis kit for RT-PCR (Roche Diagnostics, Meylan, France), according to the manufacturer's protocol. Real-time PCR was performed on a LightCycler (Roche Diagnostics) with either the LightCycler FastStart DNA Master SYBR Green 1 kit (Roche Diagnostics) or the DyNAmo Capillary SYBR Green qPCR kit (Finnzymes; Ozyme, Saint Quentin en Yvelines, France), according to the manufacturers' protocols, except that the reaction volume was reduced 2.5-fold. PCR was performed with cDNA quantity corresponding to 0.1 mm of OMCD. No DNA was detectable in samples that did not undergo reverse transcription or in blanks run without cDNA. In each experiment, a standardization curve was made using serial dilutions of a standard cDNA stock solution made from kidney RNA. The amount of PCR product was calculated as a percentage of the standard DNA. Results (arbitrary unit per mm tubule length) were calculated as means ± SE from several animals, and data in acidotic mice are expressed as percentages ± SE of mean values in control mice. Specific primers (Supplementary Table 1) were designed using LightCycler ProbeDesign (Roche Diagnostics). (The online version of this article contains supplemental data.)

Statistics.
Significant differences in tag abundance between libraries were assessed by Monte Carlo simulation analysis (58). Comparisons between other experimental groups were performed by variance analysis followed by paired least significant difference Fisher test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Acid-base status and kidney function.
Food intake diminished during the first days of NH4Cl loading before returning to control level (Table 1). Accordingly, acidotic mice lost weight. NH4Cl loading induced metabolic acidosis (low blood pH and plasma bicarbonate concentration) and increased >10-fold ammonium excretion. The GFR remained unchanged.


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  		Table 1. Functional data in control and acidotic mice

 
In 3-day acidotic mice, sodium and urea excretion was unchanged compared with controls. Urinary excretion of chloride increased along that of ammonia. In 14-day acidotic mice, urine osmolarity and concentrations of sodium, chloride, and ammonia were unchanged compared with 3-day acidotic mice, but the urine flow rate increased twofold. Urinary excretion of aldosterone increased fourfold in acidotic mice.

OMCD transcriptomes from normal and 3-day acidotic mice.
We sequenced 20,960 tags in the acidosis SAGE library. After withdrawing duplicate ditags and linker sequences, we found that the 19,146 tags remaining corresponded to 8,847 different tags (Supplementary Table 2) and Gene Expression Omnibus (accession number GSE4729). This acidosis library is similar in size and diversity to the normal and LK mouse OMCD libraries previously generated (9).

Monte Carlo simulation revealed 255 tags differentially expressed in control and acidosis libraries (Supplementary Table 3), including 157 tags overrepresented and 98 underrepresented in acidosis. Approximately 65% of differentially represented tags were reliably identified as single functionally characterized mRNAs (Table 2). Only three tags for genes known to be related to acid-base homeostasis were overrepresented in the acidosis library: H-ATPase c subunit (Atp6v0c), an H-ATPase-associated protein (Atp6ap2), and carbonic anhydrase-2 (Car2).


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  		Table 2. Number of tags differentially represented in acidosis and control libraries

 
Comparison of OMCD transcriptomes in control, acidotic, and LK mice.
Monte Carlo simulation revealed 419 different tags differentially represented between any pair among the three OMCD SAGE libraries (control, acidosis, and LK). Clustering of these 419 tags (Fig. 1) identified a subcluster (A) of tags overrepresented in the acidotic, but not in the LK library, and a subcluster (B) of tags overrepresented in the two libraries. As expected, subcluster B contains many transcripts involved in the control of cell growth and apoptosis, but no transcript known to be involved in acid-base transport. It also contains transcripts related to vasopressin signaling and functional response (Table 3). Conversely, subcluster A contains six known partners of acid-base transport (4 subunits of H-ATPase, Car2, and aldolase), several transcripts involved in cell differentiation, including galectin3, which is overexpressed during acidosis (39), but none related to cell proliferation, and only one related to apoptosis. It also includes aldosterone-induced transcripts (Sgk, ENaC, Na-K-ATPase). Figure 1 shows that the proportions of transcripts involved in transport, differentiation, and proliferation/apoptosis are markedly different in subclusters A and B.


Figure 1
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  		Fig. 1. Clusterization of tags differentially represented in control, acidosis, or potassium depletion (LK) library. We selected all the tags that were represented at significantly different levels (P < 0.05) between control and LK libraries (n = 186), control and acidotic libraries (n = 260), and acidotic and LK libraries (n = 196). After withdrawal of duplicates, occurrence of the remaining 419 tags was compared in the 3 libraries. For this purpose, tag abundance in LK and acidosis libraries was expressed as the ratio of their occurrence in LK or acidosis library over their occurrence in the control library (for this calculation and further data transformations, tag occurrences of 0 were taken as 0.5). Data were then clusterized using Cluster software (14) freely available on the web (http://rana.lbl.gov/EisenSoftware.htm) after log base 2 transformation (which allows that 0.5 and 2 ratios, which represent twofold inhibition and twofold induction, respectively, become –1 and +1 and are symmetric at ~0) and tag and library normalization (all tag and library magnitude are close to 0). Results from Cluster data treatment were graphically visualized (left part) using TreeView software also freely available at the same web address. The expression level in acidosis and LK libraries relative to control library is presented by panel color, ranging from green for minimum expression to red for maximum expression. The color intensity represents the magnitude of the deviation from the median. The dendrogram on the left represents relationships among tag abundance in the 2 libraries. The right part of the figure shows the proportions of the different functional classes of transcripts corresponding to tags overrepresented in the acidotic but not in the LK library (A, top) and of tags overrepresented in the 2 libraries (bottom, B).

 

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  		Table 3. List of tags present at higher level in 3-day acidotic and LK libraries than in control library

 
RT-PCR analysis of selected transcripts.
To validate SAGE data, RT-PCR was performed on a selection of transcripts differentially represented in control and acidosis libraries and on a group of transcripts with similar tag occurrence in the two libraries (reporter genes). At the depth of 20,000 tags per library, differential display of tags of low abundance reaches statistical significance for ≥6-fold changes. Because many tags of functional interest are present at low occurrence in SAGE libraries (5, 9) and because <6-fold changes in transcript abundance are physiologically relevant, we also investigated by RT-PCR the effect of acidosis on a selection of transcripts related to transport functions. Finally, to evaluate the time course of acidosis-induced changes in gene expression, RT-PCR was performed in 3-day and 14-day acidotic mice. RT-PCR (Table 5) confirmed the constant expression during acidosis of the four reporter genes tested, and the induction/repression of 21/23 (>90%) selected transcripts with statistically differentially represented tags in the acidosis library.

Tags of 16/21 H-ATPase subunit isoforms and one associated protein were detected in the SAGE libraries; their cumulative occurrence increased 1.9-fold at 3-day acidosis. RT-PCR demonstrated mRNA expression of 19/21 H-ATPase subunit isoforms and their progressive 1.3- to 4-fold overexpression at 3-day and 14-day acidosis (except H-ATPase A1 and D subunits, which were induced 5- to 30-fold). Among the three Cl–/HCO3– exchangers described in collecting ducts (Slc4a1, Slc26a4, and Slc26a7), only the kAE1 (Slc4a1) tag was detected by SAGE. RT-PCR confirmed in acidotic mice the kAE1 overexpression previously reported in acidotic rats (23); it revealed the expression of pendrin in control mice and its 70% inhibition in 3-day acidotic mice, whereas, in contrast to a previous report (37), Slc26a7 mRNA remained undetectable. Expression of Clcka and Clckb chloride channels, as estimated by their tag abundance, was low. Nonetheless, RT-PCR demonstrated clear-cut overexpression of Clcka already at 3-day acidosis and a smaller and delayed overexpression of Clckb at 14-day acidosis. Tags for the NH4+/NH3 carriers Rhbg and Rhcg were present at high levels in the control library. RT-PCR demonstrated marked overexpression of Rhcg during acidosis and a smaller overexpression of Rhbg only detected at 14-day acidosis. Tags for {alpha}-subunits of gastric and nongastric H-K-ATPases were absent from SAGE libraries, indicating low expression levels. However, RT-PCR demonstrated their basal expression in control mice, the marked, but transient overexpression of gastric H-K-ATPase in acidosis, and the moderate, but long-lasting overexpression of nongastric H-K-ATPase. Finally, SAGE demonstrated overexpression of Car2 and Car15, which was confirmed by RT-PCR. Although their respective tags were not found in SAGE libraries, we checked by RT-PCR for the expression of other carbonic anhydrases expressed in the kidney. Car4 and Car14 were not detected in any group. Conversely, Car12 and Car13 were detected, and moderate overexpression of Car12 was observed at 14-day acidosis.

With regard to sodium, potassium, and water transport, RT-PCR demonstrated increased mRNA levels of Sgk, vasopressin V2 receptor, protein kinase A catalytic subunit, {alpha}-subunits of epithelial Na channels (ENaC) and Na-K-ATPase, renal outer medullary potassium channel, and aquaporin (Aqp) 2 and 3 at both 3-day and 14-day acidosis. Expression of other subunits of ENaC mRNAs was not changed, but the ß3- and {gamma}-subunits of Na-K-ATPase mRNAs were increased during acidosis.

RT-PCR revealed different stimulation time courses for transcripts related to cell differentiation. Some transcripts (Grina, Itmb2, Lcn2, and Lgals3) were overexpressed with the same time course as those related to acid-base transport, whereas others were already maximally stimulated at 3-day acidosis (Gata3, Cldn4, Timp3, and Col18a1).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Control of acid-base balance.
The low expression level of pendrin (no tag detected) and its suppression during acidosis (RT-PCR) are consistent with the finding that mouse OMCDs contain no, or only a small number of, type B intercalated cells and of non-A non-B intercalated cells (47). Thus all the data from this study dealing with acid-base transport are expected to concern mainly type A intercalated cells (AICs).

Collecting duct adaptation to acute metabolic acidosis mainly results from translocation of a subapical pool of H-ATPase to the apical membrane of AICs (55). Adaptation to chronic metabolic acidosis involves two synergistic mechanisms: upregulation of acid-base transporters in AICs and an enlargement of AIC population (1). Consistent with this last adaptative mechanism, also observed during potassium depletion (9), is the overexpression of many transcripts involved in cell growth and apoptosis under the two conditions (Table 4), and the demonstration of proliferation of AICs in OMCD during acidosis (not shown). Because we do not know yet whether these genes are overexpressed in principal and/or intercalated cells, the following discussion is restricted to the expression of acid-base transporters.


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  		Table 4. List of tags present at higher level in 3-day acidotic than in either control or LK library

 
Increased activity of H-ATPase in AICs is a cardinal feature of kidney adaptation to acidosis, because H-ATPase not only generates free and titratable urine acidity but also allows bicarbonate reabsorption and ammonia secretion. Curiously, the literature regarding the regulation of H-ATPase expression during chronic metabolic acidosis is limited to the E (4, 17) and a4 subunits (45) and is contradictory, owing perhaps to different animal models or methodologies. This study demonstrates expression in mouse OMCD of mRNAs encoding all kidney-specific (B1, C2, G3, a4) and ubiquitous isoforms of H-ATPase subunits, except the E2 and G2 subunits, as well as the Vo-associated protein 2. Tag abundance of the different subunits in the control library varied from 0 to 13, confirming striking differences in the relative transcript levels that are not related to the subunits stochiometry of the H-ATPase complex (24). Metabolic acidosis induced a coordinated, time-dependent increase in mRNA level of all H-ATPase subunits expressed in the OMCD.

The data also suggest that metabolic acidosis not only modulates H-ATPase expression at the mRNA level but may also control it posttranslationally through induction of the glycolytic enzyme aldolase. As a matter of fact, aldolase directly binds H-ATPase subunits a, B, and E (31, 32) and controls H-ATPase assembly and membrane expression in yeast (32). H-ATPase also interacts with actin through its B1, B2, and C subunits (22, 54), and these interactions are crucial for the insertion of the pump into the plasma membrane (54). Thus induction of actin (actg) and of the actin monomer sequestering protein thymosin-ß4 (Tmsb4x) specifically observed in response to acidosis (Table 4) may also be involved in the control of H-ATPase activity in acidosis. Grina, Itmb2, Lcn2, and Lgals3, which are overexpressed with the same time course as H-ATPase (Table 5), may also contribute to acid-base homeostasis.


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  		Table 5. Tag occurrence and expression of a selection of transcripts in OMCD of C and A mice

 
Reabsorption of bicarbonate by OMCD also relies on the activity of cytoplasmic and apical carbonic anhydrases (44, 49) and of basolateral Cl–/HCO3– exchanger and chloride channel. Loss-of-function mutations of the cytoplasmic Car2 induce distal acidosis (36), demonstrating its nonredundant role in collecting duct acid-base transport. In contrast, the apical ecto-isoform has not been characterized yet, even though Car4 is a candidate (48). Present results support the role of Car2 in bicarbonate reabsorption, since it is expressed at a high level in the collecting duct and is induced concomitantly with H-ATPase during acidosis. Conversely, we obtained no indication as to the identity of the basolateral enzyme. Among the five other carbonic anhydrase isoforms described in the kidney, Car4 was not detected in mouse OMCDs (Table 5), Car2 and Car13 are cytosolic (29), Car12 is expressed at the basolateral border of intercalated cells (27), and Car15 is a glycosylphosphatidylinositol-anchored isoform (21), of as-yet unknown cellular origin and subcellular localization, and might be a candidate. However, the involvement of Car15 in acid-base homeostasis is unlikely because 1) it is also overexpressed in potassium depletion (Table 3) and 2) the amplitude and time course of its overexpression are different from those of other acid-base transporters (Table 5). More likely, Car15 participates in the control of cell proliferation in both acidosis and potassium depletion through local modulation of pH, as previously established for Car9 in renal clear cell carcinoma (13).

Interestingly, the mRNA levels of kAE1 and Clcka increased in parallel with H-ATPase and Car2 during acidosis, supporting the role of Clcka in the basolateral recycling of chloride accumulated in AICs by kAE1. Altogether, these data demonstrate the co-regulation of the expression level of >25 transcripts (H-ATPase subunits, kAE1, Clcka, Car2, and aldolase), which may all contribute to acid secretion in AICs and, therefore, are considered as an acid-base regulon.

Genes possibly involved in acid-base homeostasis.
Although it remains controversial whether they mediate electrogenic ammonium transport or electroneutral ammonium/proton exchange, there is a consensus for Rhbg and Rhcg being ammonium transporters expressed in the basolateral and apical membrane of OMCD AICs, respectively, which may participate in ammonium secretion (38, 57). The present finding of marked mRNA overexpression of Rhcg and weaker overexpression of Rhbg, is consistent with the recent report showing overexpression of Rhcg, but not Rhbg, in the OMCD of 7-day acidotic rats (41). The lack of overexpression of Rhbg in OMCD of 3-day acidotic mice, when ammonium excretion was markedly increased (Table 1), suggests that Rhbg is not essential for NH4+ secretion. This is consistent with the finding that Rhbg gene disruption does not impair ammonium excretion in mice, even during metabolic acidosis (6). Because ammonium induces hypertrophy of renal epithelial cells (18, 20) and because Rhcg mRNAs are overexpressed in both acidosis and potassium depletion, we propose that this overexpression may participate in the cell hypertrophy observed in acidosis (19) rather than ammonium excretion.

Our previous results in mice (12) and in rats (28) suggest that gastric and nongastric H-K-ATPases are expressed in intercalated and principal cells of the collecting duct, respectively, where they might participate in proton secretion and potassium reabsorption. However, knockout mice for either gastric or nongastric H-K-ATPase {alpha}-subunit display no major renal phenotype (34, 42), suggesting that these ATPases are not essential for acid-base and potassium homeostasis. Under these conditions, the functional significance of the marked and transient increase in gastric H-K-ATPase mRNA and the slower increase in nongastric H-K-ATPase mRNA remains unknown. Along with Car15 and Rhcg, they may also participate in the control of cell proliferation and hypertrophy through the local control of pH.

Sodium, potassium, and water handling.
Urinary sodium excretion averaged 42% and 76% of its ingested load in control and acidotic mice, respectively (Table 1). Because mice must display sodium balance close to zero within the time course of the experiment, the intestinal absorption of sodium likely increases from {approx}42% in control mice1 to {approx}76% during acidosis. Increased intestinal absorption has previously been reported during acidosis (7, 33).

Although the overall renal handling of sodium is similar in normal and acidotic mice, there are differences in the contribution of the different nephron segments to sodium reabsorption. Acidosis reduces volume reabsorption in proximal tubules (10) and consequently increases volume delivery to the macula densa and stimulates the renin-angiotensin-aldosterone system, consistently with the increased urinary excretion of aldosterone observed in acidotic mice (Table 1). In turn, aldosterone increases sodium reabsorption along collecting ducts to balance the proximal tubule defect. Along with the stimulation of the aldosterone pathway, we observed marked overexpression of sgk1 in OMCD of acidotic mice. As previously reported in response to hyperaldosteronemia in the rat collecting duct (16), we observed increased mRNA levels for the {alpha}-subunits of ENaC and of Na-K-ATPase, but not for ENaC ß- and {gamma}-subunits and Na-K-ATPase ß1-subunit.

The increased vasopressinemia previously reported in metabolic acidosis (2) likely accounts for increased Aqp2 mRNA level. The concomitant upregulation of Aqp2 and Aqp3 mRNAs may cooperate to increase water reabsorption along collecting ducts through increased expression of their cognate proteins at the apical and basolateral cell border, respectively. Increased water reabsorption along the collecting duct of acidotic mice is necessary to maintain normal urine flow rate and osmolarity, despite decreased proximal volume absorption.

Mechanism underlying changes in mRNA abundance.
Increased mRNA level may result from activation of transcription and/or from mRNA stabilization. Acidosis is able to promote the two mechanisms: in proximal tubules, it increases the abundance of phosphoenolpyruvate carboxykinase mRNA through activation of gene transcription, whereas it increases glutaminase through stabilization of its mRNAs (11). Stabilization of glutaminase mRNA is achieved through acidosis-induced binding of the {zeta}-crystallin/NADH quinone reductase complex to an adenylate and uridylate-rich element (ARE) present in its 3'-untranslated region (3'-UTR) (46). AREs are present in the 3'-UTR of many transcripts that have a short half-life and have been identified by their capacity to target the host mRNA for rapid degradation (8). This rapid degradation, which starts with mRNA deadenylation, can be prevented by ARE-binding proteins, such as the {zeta}-crystallin/NADH quinone reductase complex, in the case of glutaminase.

We have not addressed directly the mechanism of acidosis-induced increases in mRNA level, but several findings indicate that the two mechanisms may be at work in OMCDs, as in proximal tubules. On the one hand, Aqp2 mRNA level is likely controlled at the transcription level. Indeed, Gata3, whose mRNAs are increased during acidosis, binds to the GATA motifs present in the 5'-flanking region of Aqp2 gene and activates its promoter (51). On the other hand, we found that 14/55 transcripts tested by RT-PCR (Table 5), including Gata3, contain AREs (Table 6). In addition, nucleolin, known as an ARE-binding protein (40), is induced at the mRNA level in acidosis (Table 3). Two other RNA binding proteins (Rbm3 and Snrpb) overexpressed in acidosis (Table 4) are putative ARE-binding proteins.


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  		Table 6. Adenylate and uridylate-rich elements in acidosis-induced transcripts

 
To conclude, this study demonstrates the pleiotropy of the collecting ducts' response to metabolic acidosis. The comparison of the adaptative changes to distinct stimuli (acidosis vs. K depletion) furthers identification of a set of transcripts that undergo specific co-regulation. Robust changes were consistently observed during acute and chronic acidosis for dozens of mRNAs encoding proteins responsible for acid-base transport in collecting duct AICs. We propose to refer to this transcript cluster as the collecting duct acid-base regulon.


   FOOTNOTES
 
Address for reprint requests and other correspondence: A. Doucet, UMR 7134, Institut des Cordeliers, 15 rue de l'Ecole de Médecine, 75270 Paris cedex 6, France (e-mail alain.doucet{at}bhdc.jussieu.fr)

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

1 This value is much lower than that described in the rat. Although one cannot exclude the possibility that this is in part accounted for by an underestimation of urine sodium excretion secondary to incomplete urine collection in metabolic cages, it is most likely due to the fact that intestinal absorption is proportionally lower in mice than in rats. In support of this hypothesis, it should be stressed that the food ingestion/body weight and fecal mass/body weight are much higher in mice that in rats. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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M. Nowik, M. R. Lecca, A. Velic, H. Rehrauer, A. W. Brandli, and C. A. Wagner
Genome-wide gene expression profiling reveals renal genes regulated during metabolic acidosis
Physiol Genomics, February 19, 2008; 32(3): 322 - 334.
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