Abstract
ABSTRACT. Chronic metabolic acidosis enhances the ability of the medullary thick ascending limb (MTAL) to absorb NH4+ at least in part by stimulating the mRNA and protein expression of BSC1/NKCC2, the MTAL apical Na+-K+(NH4+)-2Cl− co-transporter. For assessing the mechanism by which an acid pH enhances the BSC1 mRNA abundance, MTAL were harvested from adrenalectomized rats and incubated in control (pH 7.35) and acid (pH 7.10) 1:1 mixtures of Ham’s nutrient mixture F-12 and DME. rBSC1 mRNA abundance and gene transcription rate were quantified by quantitative reverse transcription–PCR and run-off assay, respectively. Acid incubation enhanced mRNA abundance within 4 h in whole cell (P < 0.02) but not in nucleus. BSC1 gene transcription rate was not affected by acid incubation. In contrast, under conditions in which gene transcription was blocked, rBSC1 mRNA decreased within 6 h by 38 ± 11% in control but only by 15 ± 15% in acid medium (P < 0.02), which represented an increase in the BSC1 mRNA half-life from approximately 7 to approximately 17 h. Furthermore, in a mouse TAL cell line, acid incubation for 16 h significantly increased (P < 0.02) the amount of BSC1 mRNA in cells transfected with the full-length mBSC1 cDNA but not in cells transfected with a mBSC1 cDNA lacking the 3′-UTR. These results demonstrate that acid pH enhances the stability of BSC1 mRNA probably by activating pathways that act on the AU-rich 3′-UTR of BSC1 mRNA, which contributes to the renal response to metabolic acidosis. E-mail: bichara@bichat.inserm.fr
Increased urinary NH4+ excretion, which augments acid excretion, has long been recognized to be quantitatively the major compensatory response of the kidney against chronic metabolic acidosis (CMA) (1). It is established that most of ammonia that leaves the proximal tubule is absorbed by the medullary thick ascending limb (MTAL), which causes ammonia accumulation in the medullary interstitium followed by its secretion in adjacent medullary collecting tubule (2). The role of MTAL ammonia absorption may be particularly important in states of CMA because micropuncture experiments have shown that the NH4+ amount absorbed by the loop of Henle is increased under the latter condition (3,4⇓). In this regard, Good (5) has shown that rat MTAL isolated and perfused in vitro have an increased ability to absorb NH4+ in response to CMA. This adaptive response in MTAL would substantially augment NH4+ excretion during metabolic acidosis.
It is established that the luminal step of MTAL NH4+ absorption largely involves the MTAL bumetanide-sensitive Na+-K+(NH4+)-2Cl− apical co-transporter (6–10⇓⇓⇓⇓) that was recently cloned in various species and named BSC1 or NKCC2 (11–15⇓⇓⇓⇓). We recently demonstrated that CMA upregulate BSC1 expression. Indeed, BSC1 mRNA abundance significantly increased 3 h after the creation of metabolic acidosis by peritoneal dialysis, which was followed within 24 h by an augmentation of the BSC1 protein abundance (16). The augmentation of both mRNA and protein abundance persisted after 6 d of CMA induced by administration of NH4Cl in drinking water (16). Most important, we showed that incubation of freshly harvested MTAL fragments in an acid medium enhanced BSC1 mRNA and protein abundance (16). Furthermore, acid incubation stimulated BSC1 transport activity assessed in intact cells as well as in apical membrane vesicles, which was dependent on gene transcription and protein synthesis (16). These observations established acid pH as a potent regulator of BSC1 expression and explain, at least in part, the stimulation of BSC1 expression during CMA.
The present study was designed to assess the mechanism by which an acid pH augments the BSC1 mRNA abundance. To this end, we used freshly harvested rat MTAL fragments, and the results show that an acid pH increases BSC1 mRNA stability but not the BSC1 gene transcription rate.
Materials and Methods
Freshly Isolated MTAL Tubules
Suspension of Rat MTAL Tubules.
The method used to isolate MTAL fragments has been previously described in detail (17,18⇓). Because we have previously shown that glucocorticoids regulate BSC1 expression (19), MTAL fragments were harvested from rats that were adrenalectomized (ADX) and given isotonic saline as drinking water 6 d before the experiments to avoid any interference caused by endogenous glucocorticoids. All of the following steps of the preparation of the suspensions were performed under sterile conditions with use of filter-sterilized media, as described previously in detail (17).
In brief, the MTAL were washed three times by gentle centrifugation (230 × g for 2 min) and incubated at 37°C in a medium composed of a 1:1 mixture of Ham’s nutrient mixture F-12 and Dulbecco’s modified Eagle’s essential medium (HDMEM) supplemented with 5 mM heptanoic acid, 5 mM l-leucine, 15 mM HEPES, 0.1 g/L BSA, 400 UI/ml penicillin, and 200 μg/ml streptomycin. The MTAL fragments were divided into two equal parts, which gave the control and acid groups. The HDMEM media were supplemented with 10 mM Tris and 25 mM NaHCO3 (pH approximately 7.35) when gassed with 95% O2/5% CO2 for the control group or 7.5 mM Tris and 15 mM NaHCO3 (pH approximately 7.10) when gassed with 95% O2/5% CO2 for the acid group. We have previously checked by the method described below (quantitative reverse transcription–PCR [RT-PCR]) that a difference of 25 mosmol/L with mannitol at pH 7.35 had no significant effect on rBSC-1 mRNA abundance. The MTAL of each group were suspended in the HDMEM medium at the appropriate pH in 125-ml flasks placed in a rotary (100 rpm) shaking water bath at 37°C (hereafter referred to as HDMEM suspension). The HDMEM suspensions were gassed with a humidified 95% O2/5% CO2 gas mixture that was filtered through 0.45-μm filter units (Nalgene; Nalge Company, Rochester, NY) and were so maintained for several hours in the dark.
RNA Extraction, Reverse Transcription, and PCR.
MTAL total RNA was extracted from aliquots of the HDMEM suspensions with use of the SV Total RNA Isolation System (Promega, Madison, WI). The method used to obtain a competitor RNA and the quantitative RT-PCR were described previously in detail (16). In brief, a competitor RNA was obtained by a 116-bp deletion (bp 393 to 509) of the 5′ end of the rBSC1 cDNA (bp 1 to 575) in p-Bluescript by digestion with StuI and MscI restriction enzymes, and in vitro transcription of the deleted rBSC1 plasmid was performed with use of T3 RNA polymerase (mCAPTM RNA capping kit; Stratagene, La Jolla, CA). The primers used for cDNA synthesis and PCR amplification (sense, 5′-CCAAAACCAAGTGCTCGGTATT-3′ [position 107]; antisense, 5′-GGTGTTGCGGTACTCAATC [position 536]) yielded a 451-bp and a 335-bp product from the wild rBSC1 mRNA and the competitor RNA, respectively. Quantitative RT-PCR was performed with a fixed amount of MTAL total RNA and 0.27 to 4 amol of competitor RNA present together in seven tubes exactly as described previously (16). Each reaction was performed in parallel with an otherwise identical one that contained no reverse transcriptase in the reverse transcription reaction to exclude any contamination by genomic DNA. The PCR amplicons were resolved by agarose gel electrophoresis and stained with ethidium bromide, and quantification of the bands was performed by densitometry with use of the NIH Image software. After correction of the competitive DNA bands by the 451/335-bp ratio, the results are expressed in attomoles of rBSC1 mRNA/100 ng of total RNA after analysis of the linear log-log scale plot of the ratio of the fluorescence intensities of competitor RNA to MTAL total RNA. Figure 1 shows a representative example of the quantitative RT-PCR method.
Figure 1. Quantification of rBSC1 mRNA by competitive reverse transcription-PCR (RT-PCR). Digitized reproductions of agarose gels are shown in the top panel; the result on the left was obtained from 20 ng of medullary thick ascending limb (MTAL) total RNA (451 bp) and 0.5 to 4 amol competitive RNA (335 bp), and the result on the right was obtained from 80 ng of nuclear total RNA and 0.1 to 1 amol competitive RNA; RT- reaction done in the absence of reverse transcriptase. The bottom panel shows the corresponding log-log plots: the amounts of rBSC1 mRNA were 6.2 and 1.0 amol/100 ng MTAL and nuclear total RNA, respectively.
Nuclei Isolation and Run-off Assay.
Nuclei isolation and run-off assay were performed with adaptations of methods previously described by others (20). Immediately after the experiment, homogenization of MTAL fragments was performed at 4°C with use of a Dounce homogenizer (pestle B) in medium containing 10 mM Tris HCl (pH 7.5), 10 mM NaCl, 3 mM MgCl2, and 0.5% Nonidet P-40. The homogenate was filtered through a 40-μm nylon filter and centrifuged at 150 × g for 5 min. The nuclear pellet was resuspended at 4°C in storage medium containing 25% glycerol, 5 mM Tris HCl (pH 7.5), 5 mM MgCl2, 150 mM KCl, 1 mM MnCl2, 0.1 mM EDTA, 70 mM (NH4+)2SO42-, 0.2 mM AEBSF, 2.5 mM DTT, 0.1 mM CTP, 0.1 mM GTP, 0.1 mM ATP, 1 mg/ml heparin, and 260 U/ml ribonuclease inhibitor and was used immediately or stored at −80°C. For quantitative RT-PCR, nuclear RNA was extracted with the SVTotal RNA Isolation system, in which two DNAse steps are included, and then precipitated with 100 ng of yeast tRNA, 80 mM sodium acetate, and 75% (vol/vol) ethanol. Here also, each RT-PCR reaction was performed in parallel with an otherwise identical one that contained no reverse transcriptase in the reverse transcription reaction to make sure that there was no contamination by genomic DNA. Amounts of nuclear rBSC1 RNA were expressed in amol/μg total RNA as shown in Figure 1. For nuclear run-off transcription assay, 107 nuclei were incubated at 26°C for 30 min in the storage medium in which was added 100 μCi [α-32P]UTP (6000 Ci/mmol). Transcription was stopped by the addition of 100 U RNase-free DNase I followed by incubation for 15 min at 26°C. Then RNA was isolated by the phenol-chloroform extraction method with use of RNAble reagent (Eurobio) and purified by filtration through G-50 resin column (ProbeQuant G-50 Micro Columns, Amersham Biosciences). Radiolabeled transcripts (106 cpm/ml) were hybridized overnight at 70°C with a nylon membrane (Hybond-XL, Amersham Pharmacia) spotted (slot blots) with 10 μg of denatured immobilized plasmids (p-Bluescript alone and p-Bluescript containing fragments of rBSC1 and β-actin cDNA) in hybridization medium (Rapid-hyp buffer; Amersham Biosciences) supplemented with 40 μg/ml yeast tRNA, 240 μg/ml salmon sperm DNA (Stratagene), and 0.2 μg/ml polyadenylic acid (Poly A; Amersham Pharmacia Biotech). The blot was washed two times for 15 min at ambient temperature with 2× SSC, once for 10 min at 37°C with 2× SSC containing 10 μg/ml RNAsin, then three times for 15 min at 70°C in 1× SSC containing 0.1% SDS, and then exposed to autoradiography in an InstantImager (Packard). Amounts of rBSC1 nuclear RNA were calculated as specific hybridization relative to that of β-actin.
Cultured TAL Cells
Studies were performed with use of an immortalized TAL cell culture obtained from a transgenic mouse carrying the SV40 large T antigen as described previously by others (21). These cells, when grown in an HDMEM supplemented with 7% defined FBS (Hyclone, Perbio Science), 5 nM sodium selenite, and 0.03 nM insulin under a 35% O2-containing atmosphere, spontaneously express mBSC1 mRNA (21), which was confirmed in this laboratory by RT-PCR (Figure 2). Studies were performed between passages 11 and 25.
Figure 2. Digitized reproduction of a slot blot, which shows the amounts of mBSC1 and β-actin mRNA and mBSC1 and β-actin genomic and plasmid DNA in TAL cells. Transfected cells, cells transfected with mBSC1-pcDNA3. Amounts of mBSC1 mRNA and DNA in transfected cells were at least 10 and 30 times, respectively, the amounts in untransfected TAL cells.
Mutagenesis and Transient Transfection.
The full-length poly-A tail-containing 4.6-kB cDNA encoding for mBSC1-9A was excised from pSport 1 in which it was inserted (mBSC1-9A-pSport) and then subcloned into the pcDNA3 vector downstream of the human cytomegalovirus promoter/enhancer (Invitrogen, Carlsbad, CA) with use of KpnI and NotI restriction enzymes and is hereafter referred to as mBSC1-pcDNA3. For generating a vector containing mBSC1-9A lacking the 3′-UTR (approximately 1 kB) but containing the polyadenylation site and a poly-A tail (hereafter referred to as mBSC1-pcDNA3/Δ 3′-UTR), PCR amplification of mBSC1-pcDNA3 was performed with an upstream (position 3498) antisense primer (5′-TTAGGCTTTAAGAGTAAAATGTTAAG) and a downstream (position 4628) sense primer (5′-AAAATCACTACATTTTGTTTGC-TTTG).
Plated on 100-mm plastic dishes, subconfluent culture (approximately 80%) was transfected with 10 μl of lipofectamine Reagent and 6 μg of plasmids according to the manufacturer’s instructions (Invitrogen). Transfected cells were then maintained in the medium described above until confluence was achieved (1 to 2 d after transfection). Cells were then deprived from serum for 9 h and then were incubated for 16 h in an acid medium (supplemented with 12 mM HEPES, 6 mM Tris, 7.2 mM NaHCO3 [pH 7.10] under 5% CO2) or in a normal medium (supplemented with 12 mM HEPES, 8 mM Tris, 14.6 mM NaHCO3 [pH 7.35] under 5% CO2).
RNA and DNA Slot Blot Hybridization.
Total RNA was extracted from the cell lysate with use of the SV Total RNA Isolation system (Promega) and denatured for 15 min at 65°C in denaturing solution (formamide 2.5% [wt/vol], 195 mM formaldehyde, 15 mM 3-[N-morpholino]-propanesulfonic acid, 37.5 mM sodium acetate, and EDTA 7.5 mM]. Denatured RNA (5 to 10 μg) were then immobilized on a nylon membrane (slot blot; Hybond-XL; Amersham Pharmacia). Using Megaprime DNA Labeling system (Amersham Biosciences) and 17 pmol of [α-32P] CTP (3000 Ci/mmol), radiolabeled DNA probes were generated from full-length 4.6-kb mBSC1-9A cDNA (excised from mBSC1-9A-pSport) and from a 286-bp β-actin cDNA (obtained by RT-PCR from mouse TAL cell line total RNA). Prehybridization and hybridization (106 cpm/ml) were carried out for 2 h and overnight, respectively, at 65°C in Rapid-hyp buffer (Amersham Biosciences). The blot was then washed two times for 15 min at room temperature with 2× SSC with 0.1% SDS and two times for 15 min at 65°C in 1× SSC with 0.1% SDS. For DNA quantification, the cell lysate was centrifuged at 20,000 × g for 15 min, and genomic plus plasmid DNA was extracted from the pellet containing the nuclei by the phenol-Tris/chloroform method and denatured DNA (1 to 1.5 μg) was immobilized on a nylon membrane. The amounts of endogenous plus plasmid mBSC1-9A DNA and endogenous β-actin DNA were estimated by DNA hybridization with the probes described above. The hybridization and blot washing steps were performed exactly as described above for RNA slot blot hybridization. After correction for transfection efficiency estimated as the ratio of specific mBSC1-9A DNA to β-actin DNA, amounts of intact and deleted mBSC1 mRNA were calculated as specific hybridization relative to that of β-actin. As shown in Figure 2, mBSC1 mRNA was detected at a very low level in TAL cells, but transfection with mBSC1-pcDNA3 increased the amounts of mBSC1 DNA and mRNA, whereas the amounts of β-actin DNA and mRNA remained unchanged between normal and transfected cells.
Materials
[α32P]UTP and [α-32P]CTP were obtained from Amersham (Buckinghamshire, UK), and Collagenase CH grade II was obtained from Roche Diagnostics; Taq DNA polymerase, MMLV reverse transcriptase, dNTP, and yeast tRNA were obtained from Invitrogen. AEBSF, 6-dichloro-1-β-ribofuranosylbenzimidazole (DRB), and all other chemicals were obtained from Sigma-chimie S.A.R.L. (LaVerpillière, France).
Statistical Analyses
Results are expressed as means ± SEM. Statistical significance between experimental groups was assessed by paired or unpaired t test, as appropriate.
Results
Freshly Isolated MTAL Tubules
Because we have previously shown (19) that glucocorticoids regulate rBSC1 expression, experiments in the present work were performed with use of MTAL fragments harvested from rats that were ADX for 6 d. As shown in Figure 3, the abundance of rBSC1 mRNA was stable in MTAL of ADX rats, whereas it sharply decreased in MTAL of control rats, as described previously (16). This observation is consistent with the role of glucocorticoids in the regulation of rBSC1 expression.
Figure 3. Spontaneous evolution during 4 h of incubation of rBSC1 mRNA abundance in MTAL fragments harvested from control (n = 4) and adrenalectomized (ADX) rats (n = 10). Each point represents the mean ± SEM expressed in percentage of the initial value. Initial values were 10.7 ± 4.1 amol/100 ng RNAtot in control and 6.0 ± 0.5 in ADX.
Incubation for 4 h in an acid medium (pH 7.10) was sufficient to significantly increase rBSC1 mRNA abundance from 6.9 ± 1.0 amol/100 ng RNAtot in control to 8.6 ± 1.1 (P < 0.02; Figure 4). This extends results previously obtained after 16 h of incubation of MTAL harvested from normal rats (16).
Figure 4. Effect of acid pH on rBSC1 mRNA abundance measured by quantitative RT-PCR. MTAL fragments were incubated for 4 h in control (pH 7.35) and acid (pH 7.10) media. Lines connect results obtained in one experiment.
We assessed whether acid incubation enhances the rBSC1 mRNA abundance by acting on transcription by measuring the amounts of rBSC1 nuclear RNA and the rBSC1 gene transcription rate. As shown in Figure 5, rBSC1 RNA abundance in nuclei was not different between acid (5.2 ± 1.1 amol/μg total RNA) and control (5.0 ± 1.1) conditions after 6 h of incubation as assessed by quantitative RT-PCR. Figure 6 depicts the results obtained in run-off assays in which nascent transcripts are elongated in vitro in the presence of [α-32P]UTP with no new initiation of transcription, which thus assesses the rate of transcription prevailing at the time of nuclear isolation. The rate of β-actin gene transcription was not affected by acid incubation, and the relative transcription rate of rBSC1 was not different between acid and control conditions after 6 h of incubation in three independent experiments (Figure 6). These results demonstrate that acid incubation did not enhance rBSC1 mRNA abundance by stimulating rBSC1 gene transcription.
Figure 5. Abundance of rBSC1 RNA in nucleus measured by quantitative RT-PCR. MTAL fragments were incubated for 6 h in control (pH 7.35) and acid (pH 7.10) media, then nuclei were isolated and nuclear RNA was extracted as in the Materials and Methods section. Bars represent means ± SEM of results obtained in seven experiments.
Figure 6. Nuclear run-off assay. MTAL were incubated for 6 h in control or acid media, then nuclei were harvested and transcription rates were estimated by run-off assays as in the Materials and Methods section. The top panel shows the result of a representative experiment (p-Bluescript was used as a control for nonspecific hybridization). The lower panel summarizes the means ± SEM of the specific rBSC1 RNA hybridization relative to that of β-actin.
We then tested whether acid incubation enhanced rBSC1 mRNA stability. To this end, we used DRB, a specific inhibitor of RNA polymerase II. MTAL fragments were incubated for 4 h in control and acid media before adding 65 μM DRB to the suspensions followed by an additional 6 h of incubation in the presence of DRB. As shown in Figure 7, rBSC1 mRNA decay was more than twice as rapid in control as in acid medium. Indeed, rBSC1 mRNA decreased within 6 h by 38 ± 11% in control but only by 15 ± 15% in acid medium (n = 4 for each; P < 0.02). The estimated half-life of rBSC1 mRNA was approximately 7 h in control medium versus approximately 17 h in acid medium. These results demonstrate that acid incubation enhanced the stability of rBSC1 mRNA in freshly isolated MTAL fragments.
Figure 7. MTAL were incubated for 4 h in control (pH 7.35) and acid (pH 7.10) media before the addition in the media of 65 μM 6-dichloro-1-β-ribofuranosylbenzimidazole (arrow), an inhibitor of transcription. Whereas rBSC1 mRNA decreased sharply in control medium within 6 h (from 4.9 ± 1.0 amol/100 ng RNAtot to 2.9 ± 0.5), it was only slightly reduced (from 5.9 ± 1.2 to 4.8 ± 0.8) in acid medium. Points represent means (SEM are not represented to lighten the figure) of values obtained in four independent experiments. The P values analyze the differences between control and acid at each time point.
Cultured TAL Cells
Studies were performed with use of an immortalized mouse TAL cell line that spontaneously express mBSC1 mRNA when grown under a 35% O2 atmosphere (21). However, mBSC1 mRNA is expressed in these cells at a very low level compared with native kidney (21). For permitting the quantification of the mBSC1 mRNA, these cells were transfected with the pcDNA3 vector containing the full-length mBSC1-9A cDNA or the construct mBSC1-9A/Δ 3′-UTR cDNA that lacks the 3′-UTR.
As shown in Figure 8, incubation in an acid medium significantly increased the abundance of mBSC1 mRNA in TAL cells transfected with the full-length mBSC1 cDNA from 62 ± 20 in control medium to 96 ± 14 (P < 0.02) in three independent experiments. Because, as shown in Figure 2, the amount of mBSC1 mRNA in these transfected cells resulted by at least 90% from transcription of mBSC1-9A-pcDNA3 driven by a human cytomegalovirus promoter/enhancer, it could be concluded that the stimulatory effect of acid was due to stabilization of the mBSC1 mRNA, not to increased transcription. This conclusion is further supported by the lack of effect of acid on the construct mBSC1-9A/Δ 3′-UTR mRNA (Figure 8).
Figure 8. Cultured TAL cells were incubated for 16 h in control (pH 7.35) and acid (pH 7.10) media. The cells were transfected with full-length mBSC1 cDNA (BSC1-pcDNA3) or with BSC1 cDNA lacking the 3′-UTR (BSC1-pcDNA3-Δ3′-UTR). The top panel shows a digitized reproduction of a representative slot blot. The bottom panel depicts the results obtained after correction for transfection efficiency in three independent experiments. Acid increased approximately 55% the abundance of mBSC1 mRNA but not that of mBSC1-Δ3′-UTR mRNA. The abundance of β-actin was not significantly modified in these experiments.
Discussion
Present results extend the previous observation (16) that acid pH augments the rBSC1 mRNA abundance in the rat MTAL and demonstrate that this effect resulted from an increased stability of rBSC1 mRNA, not from an increased rBSC1 gene transcription rate. This result was also obtained in cultured mouse TAL cells.
We have previously demonstrated that metabolic acidosis induced by two different experimental protocols (peritoneal dialysis and administration of NH4Cl in drinking water) increased the rBSC1 mRNA and protein abundance in the MTAL (16). It is worth noting that upregulation of rBSC-1 expression by metabolic acidosis was observed independently of changes in plasma potassium concentration, in plasma osmolality, and in extracellular fluid volume (16). Furthermore, in vitro incubation of freshly harvested MTAL fragments in an acid medium increased rBSC1 mRNA and protein abundance and stimulated rBSC1 transport activity (16). However, in the latter study, MTAL were harvested from normal rats, and the abundance of rBSC1 mRNA spontaneously decreased during the first 9 h of incubation to remain stable thereafter during an additional 7 h of incubation. The effect of acid incubation to increase by approximately 58% the rBSC1 mRNA abundance was apparent only at the 16th hour of incubation, and the precise time course of the acid effect could not be determined. In the present study, we have used ADX rats to prepare the MTAL suspensions because we have previously shown that glucocorticoids enhance rBSC1 expression (19). An important observation in the present study is that rBSC1 abundance straight-away remained stable in MTAL suspensions of ADX rats, most probably because this preparation is devoid of residual vanishing effects of endogenous glucocorticoids. Under these conditions, a 4-h incubation time was sufficient for acid to enhance the rBSC1 mRNA abundance by approximately 25%, which is consistent with the observation that rBSC1 mRNA was significantly increased by 30% after 3 h of metabolic acidosis induced by peritoneal dialysis (16). The acid effect did not result from an enhanced transcription because the abundance of rBSC1 in the nucleus and the rBSC1 gene transcription rate were not affected by acid. Rather, acid enhanced the stability of rBSC1 mRNA as reflected by a much slower mRNA decay under acid than under normal condition after blockade of transcription (Figure 7). Thus, acid more than doubled the half-life of rBSC1 mRNA. Furthermore, in an immortalized mouse TAL cell line, acid incubation increased the amount of full-length mBSC1-9A mRNA in transfected cells. However, the amount of mBSC1 mRNA lacking the 3′-UTR was not increased by acid incubation. These results demonstrate that acid also enhanced the stability of mBSC1 mRNA in cultured mouse TAL cells.
The molecular mechanisms of the stabilization by acid of rBSC1 mRNA were not investigated in the present study. It must be emphasized that we first used fresh tissues to guarantee the physiologic relevance of the experimental data. That the same result was obtained in mouse TAL cells transfected with the full-length mBSC1-9A cDNA but not in cells transfected with mBSC1 cDNA lacking the 3′-UTR indicates that sequences in the 3′-UTR of mBSC1 mRNA are the targets responsible for the acid effect. It is worth noting that the 3′-UTR of the rat BSC1 mRNA contains, in an AU-rich environment, several AUUUA sequences that may behave as adenylate, uridylate-rich instability elements (Figure 9). The mouse BSC1 mRNA have the same property (seven AUUUA sequences in an AU-rich environment; not shown), which is characteristic of mRNA the stability of which is highly regulated (reviewed in (22–25⇓⇓⇓). That the stability of an mRNA may be increased by an acid pH is not unprecedented. This is the case, for example, of the mitochondrial glutaminase mRNA of the proximal tubule, which contains a direct repeat of an eight-base AU sequence that behaves as a pH-response element to which binds acid pH-activated ζ-crystallin/NADPH:quinone reductase to increase the stability of the glutaminase mRNA (reviewed in (26). Further works using cultured cells are needed to address these issues as well as that of the signal transduction pathway that is activated by an acid pH.
Figure 9. Sequence of the 3′-untranslated region common to the rat A, B, and F isoforms of BSC1 mRNA (GenBank accession number U10096). This mRNA segment contains 1042 bases with 63% A and U bases. Five AUUUA sequences are in underlined bold upper case.
In summary, present results establish for the first time that an acid pH enhances the stability of rBSC1 and mBSC1 mRNA to increase within a few hours the abundance of these mRNA in the MTAL. This mechanism may explain, at least in part, how CMA enhances rBSC1 mRNA and protein expression in the MTAL (16). An increased BSC1 expression may be the main cause of the increased ability of the MTAL to absorb NH4+. This would aid in enhancing NH4+ urinary excretion and thus would contribute to the renal response to CMA.
Acknowledgments
This study was supported by grants from the Institut National de la Recherche Médical and the Université Paris 7. Z.K. and A.A.-E. were supported by grants from the Fondation pour la Recherche Médicale Française.
We thank David B. Mount and Glenn T. Nagami for the generous gifts of the mBSC-9A-pSport cDNA and the immortalized mouse TAL cell culture, respectively. We thank Laetitia Micheli for technical help.
- © 2003 American Society of Nephrology
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