1Chair of Nephrology, Second University of Napoli, Naples; 2Prassis Research Institute, Sigma-Tau, Milan; 3Laboratory of Environmental Physiology, Grange Blanche Faculty of Medicine, Lyon Cedex, France; 4Department of Physiology, University of Tuebingen, Tuebingen, Germany; and 5Chair of Nephrology, Ateneo Vita e Salute, S. Raffaele Hospital, Milan, Italy
Submitted 21 June 2004 ; accepted in final form 25 January 2005
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ABSTRACT |
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type 3 sodium/hydrogen exchanger; sodium-potassium-2chloride cotransporter; sodium-chloride cotransporter; epithelial sodium channel; sodium; kidney
We present data concerning the transcript and protein abundance of these four sodium transporters measured in 23- to 25-day-old MHS rats and in a normotensive age-matched control strain (MNS). We confirmed that the MHS animals are not yet hypertensive and that they have an increased glomerular filtration rate (GFR) compared with MNS rats. Quantitative PCR, applied on recognized tubular segments (10, 38), and Western blot experiments reveal that there are no major difference in NHE3 expression and protein abundance between the two strains but that MHS animals show significantly increased expression, along the medullary TAL, of the NKCC2 gene at both the mRNA and protein levels. Interestingly, NCC gene transcript abundance, measured in distal convoluted tubules, and related protein abundance were significantly reduced in MHS. Finally, the abundances of mRNA and protein of -ENaC were also decreased in both the cortical and outer medullary collecting ducts in MHS, whereas
- and
-ENaC protein abundances were unaffected.
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METHODS |
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Measurement of GFR.
The animals were anesthetized [ketamine (60 mg/kg body wt) plus xylazine (10 mg/kg body wt)], and the right femoral artery was catheterized (PE-10 tubing) to record blood pressure and to obtain blood for measurements of inulin. The left femoral vein was cannulated and used for intravenous (iv) infusion via a syringe pump (Braun Apparatus) of a modified Ringer-saline solution containing inulin (0.1 mg/ml; Inutest polyfructosan; Boehringer-Mannheim, Mannheim, Germany). The two catheters were externalized through a small incision made in the skin at the back of the head. They were kept filled by flushing them with heparinized solution to prevent clotting. Four hours after the surgery, when the rats were completely recovered from anesthesia, a priming dose of inulin (0.4 ml/100 g body wt) was infused followed by a maintaining dose at 0.6 ml·h1·100 g body wt1. After an equilibration period of 60 min, three arterial blood samples (20 µl) were taken every 20 min. According to Earle and Berliner (13), under these conditions the infusion rate of inulin will equal the excretion rate and therefore the GFR may be calculated as:
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Mean arterial blood pressure was measured in conscious rats through the femoral artery catheter connected to a pressure transducer (XCDR-P33 XL, Gould, Cernusco-Milan, Italy) attached to a multichannel recorder (Gould Instrument System, RS 3800).
Plasma osmolality, AVP, and aldosterone measurements. Trunk blood was carefully collected after decapitation of the animals. Plasma osmolality was measured on 20-µl samples using a Fiske One-Ten osmometer. For plasma AVP measurement, the extraction procedure was miniaturized from that used by Skowsky et al. (42), as previously described (19), using 700-µl plasma samples, with adsorption of the hormone onto bentonite followed by an elution using acidified acetone (80% acetone-20% 1N HCl). Intra- and interassay coefficients of variation were 7 and 13.5%, respectively. AVP was then measured by radioimmunoassay using antiserum K9-IV (gift of Dr. L. C. Keil, NASA Ames Research Center, Moffett Field, CA) (25), as previously described (19), synthetic (375 IU/mg, gift of Ferring, Malmö, Sweden), and 125I-labeled iodo-AVP prepared in our laboratory. The assay has a sensitivity of 0.25 pg/assay tube, an ED50 of 1.7 pg AVP, and a useful range up to 50 pg/assay tube.
For the aldosterone measurements, the rats were anesthetized with ether, the abdomen was opened, and the blood was drawn from the abdominal aorta. Blood collection was interrupted as soon as dyspnea appeared. Plasma aldosterone concentrations were determined using a commercially available radioimmunoassay kit (ALDOCTK-2, Sorin P2714, Sorin Medica, Saluggia, Italy).
Tubule microdissection. After anesthesia [ketamine (60 mg/kg body wt) plus zylazine (10 mg/kg body wt)], the left kidney was removed and washed in ice-cold dissection solution containing (in mM) 137 NaCl, 5.4 KCl, 25 NaHCO3, 0.3 Na2HPO4, 0.4 KH2PO4, 0.5 MgCl2, 10 HEPES, 5 glucose, and 1 glycine. Proximal and distal convoluted tubules, medullary TAL, and cortical and outer medullary collecting ducts were isolated according to the method described earlier by Schafer et al. (39). Small pieces of renal cortex (for proximal tubules, distal tubules, and cortical collecting ducts) and inner stripe of outer medulla (for TAL and outer medullary collecting ducts) were cut under the stereomicroscope and incubated for 30 min at 37°C in the microdissection solution containing 0.5 mg/ml collagenase, continuously bubbled with 95% O2-5% CO2. After digestion, the tissues were washed with ice-cold collagenase-free dissection solution containing 1 g/l albumin. Proximal tubules, distal tubules, and cortical collecting ducts were isolated free hand from the renal cortex under the stereomicroscope; distal tubules were identified by their appearance among the other superficial tubules of the renal cortex. TAL were separated from other cells and fragments by filtering the supernatant through a 80-µm-opening nylon mesh (Millipore). The use of collagenase eased the tubular dissection, thus allowing harvesting of enough material for the molecular biological experiments.
RNA purification.
Total RNA (0.52 µg) was purified from proximal tubules, TAL, distal tubules, and collecting ducts. After isolation, the tubules were transferred into 350 µl of lysis buffer containing guanidinium isothiocyanate and -mercaptoethanol. The lysate material was loaded on a silica gel membrane (Qiagen) that specifically binds total RNA. Possible contamination from genomic DNA was prevented by incubation of the membrane with 30 U of RNase-free DNase I (Qiagen) for 15 min at 25°C. The membrane was washed three times, and total RNA was eluted in 50 µl of 1 g/l diethylpyrocarbonate (DEPC-water). The concentration and purity of RNA were determined by measuring its absorbance at 260 and 280 nm using a GeneQuant RNA/DNA calculator (Pharmacia Biotech).
Reverse transcription. cDNA was synthesized from equal amounts of total RNA (100200 ng) using 200 U murine leukemia virus reverse transcriptase (GIBCO BRL), 0.5 µg of oligo(dT)12-18 (GIBCO BRL), 10 mM dithiothreitol, and 2.5 mM dNTP (Pharmacia) in a total volume of 20 µl. Before the addition of reverse transcriptase, the reaction mixture was incubated at 70°C for 3 min to denature the RNA, then cDNA was synthesized at 37°C for 1 h. Controls, incubated as above, but without addition of reverse transcriptase, were included in each run.
PCR.
PCR reactions were performed in a total volume of 50 µl in the presence of: 10 pmol of each oligonucleotide primer, 200 µM dNTP solution, 5 µl of 10x PCR buffer, 1.5 mM MgCl2, and 1.25 U Taq polymerase. Primer sequences are listed in Table 1. The expected sizes of the PCR products were 622 bp for NHE3, 603 bp for NKCC2, 550 bp for NCC, and 678 bp for -ENaC. Samples were denatured at 95°C for 3 min, followed by 33 cycles consisting of denaturing at 95°C (1 min), annealing at 65, 60, 58, and 60°C (30 s) for NHE-3, NKCC2, NCC, and
-ENaC, respectively, and extension at 72°C (1 min). After amplification, PCR products were subjected to size separation by agarose gel electrophoresis (18 g/l).
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All the PCR reactions were performed as described before. The various products were analyzed by agarose gel electrophoresis (18 g/l), and they were recovered from the gel using an agarose gel DNA extraction kit (Clontech). The concentration and purity were determined by measuring the absorbance at 260 and 280 nm.
Quantification of NHE3, NKCC2, NCC, and -ENaC mRNA.
For each mRNA quantification, competitive PCR reactions were performed using as templates the cDNA obtained from total RNA, extracted from the various tubular segments, and the corresponding internal standards. The NHE3-specific primers yielded products of 622 and 546 bp for wild-type and internal standard, respectively. The NKCC2-specific primers produced fragments of 603 and 520 bp for wild-type and internal standard, respectively. Two PCR products of 550 (wild-type) and 453 bp (internal standard) were yielded by NCC-specific primers. For
-ENaC, the PCR products were of 678 and 605 bp. Five to eight competitive PCRs were performed by addition of decreasing amounts of the competitive template (internal standard) to replicate reactions containing identical amounts of cDNA. A progressive decrease in the competitive template PCR product corresponds to a progressive increase in the wild-type template PCR product. The PCR products were separated on 18-g/l agarose gels and stained by ethidium bromide. The gel was photographed, and fluorescence intensity of PCR products was quantified using NIH Image 1.60. The amounts of NHE3, NKCC2, NCC, and
-ENaC mRNA were calculated using a log-log scale plot of the ratio of PCR products vs. the known amount of internal standard used in the competitive PCR reactions. When the wild-type and competitive PCR products were equivalent [log (ratio) = 1], the amounts of wild-type NHE3, NKCC2, NCC, and
-ENaC mRNA present in the initial sample were equal to the known starting amount of the competitive template. Results are expressed per nanogram of total RNA.
Western blot analysis of NHE3, NKCC2, NCC, and ENaC subunit protein abundances. Thin pieces (100200 mg) of renal cortex (for NHE3, NCC, and ENaC subunits) and of inner stripe of outer medulla (for NKCC2 and ENaC subunits) were frozen at 80°C and then disrupted with a Potter homogenizer at 4°C in membrane buffer (150 mM NaCl, 50 mM Tris·HCl, 5 mM EDTA) containing (in µg/ml) 50 4-(2-aminoethyl)-benzenesulfonyl fluoride, 2 leupeptin, and 2 aprotinin. After the centrifugation at 10,000 g for 15 min at 4°C, the supernatant was centrifuged again at 100,000 g for 20 min. The protein concentration of the supernatant was determined by Bradford assay.
To evaluate the abundance of NHE3, NKCC2, NCC, and ENaC subunits, 30 µg of proteins were diluted in 5x loading buffer (10 g/l SDS, 10% glycerol, 1% 2-mercaptoethanol, 5 mM Tris·HCl, pH 6.8), boiled for 5 min, and separated using two 8% SDS-PAGE. After electrophoresis, the first gel was stained with Coomassie blue and the second gel was submitted to electroblotting to transfer the proteins onto a polyvinylidene difluoride membrane that was washed with PBS. After the wash, the membrane was incubated with blocking buffer (2 g/l high purified casein, 1 g/l Tween 20 in PBS) for 1 h and then probed for 1 h with the antibody. A secondary goat anti-rabbit IgG+IgM alkaline phosphatase conjugate was diluted 1:5,000 in 5 ml of blocking buffer and added for 1 h, after two washes in 20 ml of blocking buffer. The last washing was performed three times with 20 ml of blocking buffer, and finally detection was obtained with a CSPD chemiluminescent substrate (Tropix, Bedford, MA). To test for equal loading of proteins, 30 µg anti--actin antibody (Sigma, St. Louis, MO) diluted 1:1,000 or 0.5% Ponceau S in acetic acid was used. NHE-3, NKCC2, NCC,
-ENaC,
-ENaC,
-ENaC, and
-actin abundances in the various nephron segments of MNS and MHS rats were quantified by densitometric analysis.
Antibodies.
We used previously well-characterized rabbit polyclonal antibodies to the following proteins: NHE-3, NKCC2, and NCC. They were provided by Orson Moe (1), Steve Hebert (24), and David Ellison (7), respectively. For -ENaC, commercially available polyclonal rabbit antibodies were used (Sigma).
and
-ENaC antibodies were provided by Bernard Rossier (12).
Statistical analysis. Statistical analysis was performed using the unpaired t-test. All data are expressed as means ± SE.
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RESULTS |
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NHE3 protein abundance was determined by Western blot analysis using a rabbit polyclonal antibody. As shown in Fig. 1D, the antibody recognized a protein with a molecular mass between 66 and 116 kDa. Densitometric analysis (Fig. 1E) demonstrates that NHE3 abundance, normalized for -actin, was similar in the two strains (MNS: 1.12 ± 0.06, n = 7 vs. MHS: 0.90 ± 0.13, n = 7), thus confirming, at the protein level, the data of the competitive PCR.
NHE3 mRNA abundance in TAL was again similar in MNS (2.36 ± 0.31 fmol/ng total RNA, n = 8) to MHS (1.94 ± 0.31 fmol/ng total RNA, n = 8). The same was found for NHE3 protein abundance that, normalized for -actin, was 1.31 ± 0.29 in MNS (n = 5) and 1.26 ± 0.21 in MHS animals (n = 5).
NKCC2 mRNA and protein abundance along the TAL. As shown in Fig. 2C, NKCC2 mRNA abundance was increased 2.4-fold in TAL of MHS rats (2.37 ± 0.24 fmol/ng total RNA, n = 8) vs. in TAL of MNS (1.00 ± 0.11 fmol/ng total RNA, n = 8).
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NCC mRNA and protein abundance along the distal convoluted tubule. The results of the competitive PCR reactions are shown in Fig. 3, top: NCC mRNA abundance was significantly (P < 0.005) lower in distal tubules of MHS rats (0.16 ± 0.07 fmol/ng total RNA, n = 7) than in distal tubules of MNS rats (0.69 ± 0.14 fmol/ng total RNA, n = 5).
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-ENaC mRNA in collecting ducts.
The results of the competitive PCR reactions are shown in Fig. 4:
-ENaC mRNA abundance was significantly (P < 0.05) lower in the cortical collecting duct of MHS rats (1.38 ± 0.06 amol/ng total RNA, n = 5) compared with MNS rats (1.67 ± 0.11 amol/ng total RNA, n = 4) (Fig. 4C). The same holds for mRNA along the outer medullary collecting ducts (Fig. 4D): it was 1.18 ± 0.02 amol/ng total RNA, n = 4, in MHS rats and 1.71 ± 0.19 amol/ng total RNA, n = 4, in MNS animals (P < 0.05).
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DISCUSSION |
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NHE3 mRNA and protein abundance in proximal tubules. Determination of NHE3 mRNA and protein abundance in renal proximal tubules isolated from 23- to 25-day-old MHS and age-matched MNS rats reveals that, in the prehypertensive phase, NHE3 transcript and protein abundances are not significant different in the two strains of rats, thus indicating that the modulation of gene expression does not seem to be involved in the induction of hypertension in the MHS animals. These results are in seeming contrast to a previous report demonstrating that Na+/H+ exchanger activity is increased in MHS compared with MNS rats (21). Since in the present study we have actually measured the expression and the abundance, but not the activity, of NHE3, it cannot be excluded that the contribution of the NHE3 gene to MHS hypertension is mediated by its increased activity and not necessarily associated with an overexpression. Moreover, although NHE3 has been identified as the most abundant isoform of the Na+/H+ exchanger present in the apical membrane of the proximal tubule and involved in trans-epithelial sodium reabsorption, several reports indicate that other isoforms of the Na+/H+ exchanger family, some of which are yet unidentified, may be involved in sodium transport (46). It should be stressed that the role and, more importantly, the mechanism involving NHE3 in the pathogenesis of genetic hypertension are still elusive. For example, in studies in spontaneously hypertensive rats (SHR) there are reports that indicate that NHE3 is regulated at the transcriptional level (26), whereas others suggest that a posttranscriptional mechanism is responsible for the overexpression of NHE3 protein and the associated increase in Na+/H+ exchanger activity (27). Finally, the redistribution of NHE3 in proximal tubule cells may be an additionally important factor in understanding sodium transport along the proximal tubule during acute and chronic hypertension (49).
NKCC2 mRNA and protein abundance along the TAL.
One of the new findings of the present study is the identification of NKCC2 as a potential player in the induction of this form of genetic hypertension. Indeed, as shown in Fig. 2, NKCC2 gene expression, measured in the medullary TAL, is significantly enhanced at both the mRNA and protein level in MHS compared with MNS rats. It is also noteworthy to underline that the 2.4-fold increase in mRNA is not substantially different from the 2.1-fold increase in NKCC2 protein amount, even though this last value was corrected for -actin abundance. These results are in excellent agreement with the previous report from Ferrandi et al. (15) demonstrating that NKCC2 activity (measured as bumetanide-sensitive 86Rb uptake) in membrane vesicles isolated from the same zone of the kidney, i.e., the outer medulla, is significantly increased in MHS compared with MNS rats (15). Moreover, they fit very well with another report from the same group demonstrating a major rise in the abundance and activity of the Na+-K+-ATPase in the outer medulla of MHS animals (16).
The strong stimulation of NKCC2 could potentially contribute to the significant rise in blood pressure. Several lines of evidence support this hypothesis. First of all, the TAL is an important site of sodium transport; actually, roughly 20% of the filtered sodium load is normally reabsorbed at this level. Second, apical sodium entry in the TAL is mainly mediated by NKCC2 (18) as demonstrated by the copious natriuresis associated with the use of loop diuretics, an effect that is enhanced by their inhibiting action on TGF (48). Third, patients carrying a mutation with a loss of function of the NKCC2 gene (type I Bartter's syndrome) are characterized by orthostatic hypotension (8). Finally, mice lacking the NKCC2 gene suffer from severe salt wasting resulting in rapid death (44). It is noteworthy to underline that upregulation of NKCC2 has been also reported in the early phase of prenatally programmed hypertension induced by a maternal low-protein diet during pregnancy (30).
The stimulation of NKCC2 may also be involved in the increased GFR found in the MHS animals. In fact, activation of ion transport along the TAL is expected to decrease the delivery of sodium chloride to the macula densa, thus enhancing GFR through the tubuloglomerular feedback mechanism. In young MHS rats, the alteration of TGF, together with an increase in net interstitial pressure, has already been described by Boberg and Persson (6), whereas in the present paper we have probably identified the molecular mechanism involved in this process.
NCC mRNA and protein abundance in the distal convoluted tubule. Our findings further indicate that NCC gene transcript and protein abundances along the distal convoluted tubule are significantly decreased in MHS rats compared with MNS (Fig. 3).This effect may be related to decreased sodium load that reaches the distal tubule due to the larger trans-epithelial sodium transport along the TAL linked to the overexpression of NKCC2. Indeed, it has been demonstrated that high rates of ion delivery to the distal nephron, following chronic administration of furosemide, raised [3H]metolazone binding sites (an index of NCC function) in membranes extracted from renal cortex (11) and enhanced NCC transcript levels in distal tubular cells (35). Although in the classic "escape" phenomenon either blood pressure is raised or aldosterone levels are clamped high, it is possible that the decrease in NCC abundance may be related to the "escape" phenomenon, as it has been demonstrated in animal models (47). Such a hypothesis is supported by three observations: first, the unchanged aldosterone plasma levels indicate that this phenomenon is independent from aldosterone-mediated gene regulation; second, at this age, the MHS rats are in a state of a slight volume expansion (4) and the decrease in the abundance of NCC may be necessary to restore sodium balance; and third, the young MHS rats exhibited a significantly higher net interstitial pressure than the age-matched MNS (6). This last finding may indicate that NCC is the key molecular target of interstitial pressure variations as it has been suggested in animal models of long-term regulation of pressure natriuresis (29).
The likelihood that NCC may be involved in the pathogenesis of genetic hypertension has been forwarded by Beaumont et al. (3), who found that the thiazide receptors in the renal cortex in SHR increased with age together with the development of hypertension. However, Moreno et al. (34) have recently demonstrated that NCC transcript levels did not change in three models of experimental hypertension (including SHR). Therefore, to explain the reported increase in thiazide receptor density, other NCC functions must be envisaged such as stability, protein translation rate, and intracellular distribution.
ENaC subunits in the collecting ducts.
In the present study, we have also examined the expression of ENaC that is the key apical Na+ transporter from the connecting tubule to the collecting duct. ENaC is a heteromultimeric channel, composed of three homologous -,
-, and
-subunits (9). It has been shown that
-ENaC is mostly present at the apical domains of the principal cells, whereas
- and
-ENaC are mainly localized in cytosolic vesicles (20). The significance of this different subcellular distribution of the three subunits has not been ascertained. Oocyte injection experiments demonstrate that the
-subunit is essential for the function of the channel but that the channel activity is enhanced by association with
- and
-subunits (2), a finding that has been confirmed by the generation of gene knockout mice for the individual subunits (23). Finally, aldosterone and vasopressin regulate ENaC-dependent sodium reabsorption by changing the expression of the individual ENaC subunits (14, 32). In accordance with the results obtained for NCC, our present findings indicate that both in the cortex and in outer medulla of MHS kidneys there is a modest, although significant, downregulation of
-ENaC. Since apical Na+ entry is believed to constitute the rate-limiting step in Na+ reabsorption in the different nephron segments, it is likely that decreased expression of apical membrane Na+ carriers reflects inhibition of transepithelial Na+ transport. It is therefore obvious to postulate that in this model of sodium-dependent hypertension, the early phase is characterized by decreased Na+ transport in the tubular segments beyond the TAL. Such a phenomenon is not unique to this model, because in the prenatally programmed hypertension the vigorous upregulation of NKCC2 is partially compensated for by a slight reduction of
-ENaC (30). With respect to the
- and
-subunits, the lack of changes is not surprising because, in other experimental models associated with changes in renal sodium handling, the regulation of the channel may be dependent on other factors like a degradation of the
-subunit (43). We must emphasize that with our technical approach we are able to detect only changes in channel protein abundance, whereas epithelial sodium reabsorption via ENaC may be controlled in addition by changing the conductance or the gating kinetics of functional channels.
Cellular and hormonal mediators. The important finding of our study is the increased NKCC2 gene expression in MHS rats. Several hypotheses may be advanced to identify the signaling mechanism(s) responsible for this effect. The first possible mechanism is superoxide. Recently, it has been demonstrated that the TAL is the major source of superoxide production (28); in addition, superoxide stimulates sodium chloride absorption along the TAL (36), and our preliminary data indicate that the kidney of MHS generates more superoxide compared with MNS animals (Capasso G, Cantone A, and Poulet R, unpublished observations). All these studies may indicate a role of superoxide in the modulation of the function of NKCC2. Second, it could be related to morphological changes in the kidney and tubular cells; in fact, it has been demonstrated that the size of the kidneys and the volume of the tubular cells in MHS are supposed to be smaller than MNS rats (17). This would necessitate a higher GFR-to-kidney size ratio to filter the blood; it is therefore reasonable to postulate that the increased single-nephron sodium load may trigger the reported upregulation of TAL sodium reabsorption. These findings are quite intriguing, because in rats with low birth weight induced by a maternal low-protein diet, the development of hypertension has been related to transcriptional upregulation of NKCC2 (30). Finally, it is still possible that enhanced expression of NKCC2 in MHS may be dependent on aldosterone and/or vasopressin (ADH). However, we have not found any statistical difference in aldosterone levels between MNS and MHS, thus confirming previous results from Mantero et al. (31); the same holds for ADH plasma levels between the two strains of rats (Table 2). Although measurements of these hormones in young rats are extremely difficult and the results are largely variable, it looks as if aldosterone and ADH may not be the possible causes of the increased expression of NKCC2.
Conclusions.
We have found that in young MHS animals, characterized by increased GFR and a still normal blood pressure, the expression and abundance of NHE3, measured along the proximal tubule and the TAL, are not different, whereas the NKCC2 transporter, expressed along the medullary TAL, is upregulated compared with in age-matched MNS animals. Along the distal convoluted tubule, NCC gene transcript and protein abundances are significantly reduced in MHS. Finally, in the collecting ducts there is downregulation of gene expression and protein abundance of -ENaC. Therefore, the upregulation of NKCC2 along the medullary TAL does not induce an increase in systemic blood pressure, probably because its action on salt reabsorption is offset by the downregulation of NCC and
-ENaC, and by the concomitant increase in GFR.
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GRANTS |
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ACKNOWLEDGMENTS |
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Part of this work was presented at the 2002 American Society of Nephrology meeting and published in abstract form (J Am Soc Nephrol 13: A3072, 2002).
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FOOTNOTES |
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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.
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REFERENCES |
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