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1The Water and Salt Research Center, 2Institute of Clinical Medicine, and 5Department of Cell Biology, Institute of Anatomy, University of Aarhus, Aarhus C; 3Department of Paediatric Surgery, 3rd Teaching Hospital and Institute of Clinical Medicine, Zhengzhou University, Henan, China; 4Department of Internal Medicine, Chonnam National University Medical School, Gwangju, Korea; 6Department of Clinical Physiology, Aarhus University Hospital-Skejby, Aarhus N; and 7Department of Nephrology, Aarhus University Hospital-Aalborg, Aarhus, Denmark
Submitted 10 September 2007 ; accepted in final form 23 May 2008
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ureteral obstruction; HCO3– reabsorption; urinary acidification defect
The kidney maintains systemic acid-base homeostasis via two processes (15): 1) reabsorption of filtered HCO3–, which mainly occurs in the proximal convoluted tubule; and 2) excretion of acid through the titration of urinary buffers and the excretion of ammonium, which takes place primarily in the distal nephron. In both processes, a variety of acid-base transporters are involved (15). The proximal tubule of the mammalian kidney reabsorbs >80% of the filtered bicarbonate (33, 36) by the apically expressed type 3 Na+/H+ exchanger (NHE3) in conjunction with the basolaterally expressed electrogenic Na+/HCO3– cotransporter (NBC1). Moreover, studies using NHE3 null mice demonstrated that HCO3– reabsorption in the proximal tubule decreased up to 60%, consistent with the functional importance of NHE3 in HCO3– reabsorption (35). The SLC4A4 gene encodes for the Na+/HCO3– cotransporter (NBC1), and mutations in SLC4A4 cause permanent, isolated proximal renal tubular acidosis (8, 16, 17).
In the thick ascending limb (TAL) of Henle's loop, the electroneutral Na+/HCO3– cotransporter, NBCn1, which is localized in the basolateral plasma membrane of TAL cells in the outer medulla (7, 39), may participate in HCO3– transport into cells to maintain intracellular pH levels (23). Moreover, the apical bumetanide-sensitive Na+-K+-2Cl– cotransporter NKCC2, a major apically expressed protein for sodium reabsorption in the TAL (29), previously was demonstrated to mediate NH4+ absorption by replacing K+ (18, 43).
The collecting duct (CD) is the final site for urine acid-base regulation. The collecting duct is composed of a "tight" epithelium that can generate steep pH gradients and thus plays a major role in the excretion of net acid (28, 33, 36). Two main types of intercalated cells, type A and type B (1, 5, 15, 40), can be distinguished on the basis of both the morphological characteristics and the subcellular localization of acid-base transporters. Type A intercalated cells secrete H+ into the urine via a vacuolar H+-ATPase that is located to the apical plasma membrane and apical vesicles and reabsorb HCO3– to the blood through band 3-like Cl–/HCO3– exchanger (AE1) localized to the basolateral plasma membrane (1, 5, 15, 19, 40). Type B intercalated cells secrete HCO3– in exchange for Cl– across the apical membrane through the novel anion exchanger pendrin (15, 21, 34, 40), whereas protons are secreted to the systemic circulation by the vacuolar type H+-ATPase localized in the basolateral membrane. Recent studies have demonstrated an important role of pendrin in disorders of acid-base metabolism (10).
The present study therefore aimed at examining the changes in protein expression of key renal acid-base transporters in the different nephron segments in response to bilateral ureteral obstruction (BUO) and release of the obstruction to identify the underlying cellular and molecular mechanisms for the urinary acidification defect associated with ureteral obstruction.
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METHODS |
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All procedures conformed with the Danish national guidelines for the care and handling of animals and to the published guidelines from the National Institutes of Health. The animal protocols were approved by the board of the Institute of Clinical Medicine, University of Aarhus, according to the licenses for use of experimental animals issued by the Danish Ministry of Justice. Studies were performed using male Munich-Wistar rats, initially weighing 250 g (Møllegard Breeding Centre, Eiby, Denmark). Rats were maintained on a standard rodent diet (Altromin, Lage, Germany) with free access to water. Rats were assigned randomly to either a sham-operated control or experimental groups and kept in individual metabolic cages, with a 12:12-h artificial light-dark cycle, a temperature of 21 ± 2°C, and humidity of 55 ± 2%. Rats were allowed to acclimatize to the cages for 3 days before surgery. Water intake, urine output, and body weight of the rats were monitored every 24 h during the study.
Rats were anesthetized with isofluran (Abbott Scandinavia), and during surgery the rats were placed on a heated table to maintain constant temperature (rectal temperature at 37–38°C). In protocol 1, through a midline abdominal incision, both ureters were occluded by silk ligature for 24 h, and then the rats were killed. In protocol 2, the ureters were exposed and a 4-mm-long piece of bisected polyethylene tubing (PE-50) was placed around the midportion of each ureter (12, 25, 26). The ureter was then occluded by tightening the tubing with a 4-0 silk ligature. After surgery, the rats were allowed to regain consciousness. Twenty-four hours later, rats were anesthetized again and the obstructed ureters were decompressed by removal of the ligature and the PE tubing. With this technique, the ureters could be completely occluded for 24 h without evidence of impaired ureter function. Rats were randomly allocated to the protocols indicated below. Age- and time-matched, sham-operated controls were prepared and were observed in parallel with each experimental group in the following protocols (Fig. 1).
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Protocol 2. BUO was induced for 24 h, followed by release, and rats were observed for the following 4 days (n = 19). To examine whether release of BUO (BUO-R) was associated with an acidification defect, BUO-R rats were subjected to either vehicle (BUO-R; 3 ml of water, n = 11) or acid loading (BUO-A; 0.033 mmol NH4Cl/g body wt in 3 ml of water, n = 8) (10, 14) by gavage once a day during the last 2 days before termination. In parallel, sham-operated animals were also subjected to acid loading (Sham-A; n = 12) or vehicle (Sham; n = 7). Semiquantitative immunoblotting was performed in BUO-R and Sham groups.
Clearance Studies
Body weight, water intake, food intake, and urine output were observed during the time rats were maintained in the metabolic cages. Urine was collected during 24-h periods throughout the study.
Clearance studies were performed during the last 24 h in each protocol. During anesthesia, at the end of each protocol, 3–4 ml of arterial blood was drawn in gas-tight syringes from the abdominal aorta before removal of the kidneys. One aliquot of the blood sample was used immediately for blood-gas analysis in an automatic blood-gas analyzer (ABL system 615, Radiometer, Copenhagen, Denmark), providing pH, plasma total CO2, standard bicarbonate and base excess, and plasma concentration of chloride in the arterial blood. The remaining blood was centrifuged for the determination of plasma electrolytes and osmolality. The plasma concentrations of creatinine, urea, sodium, and potassium were determined (Kodak Ektachem 700XRC). The osmolality of urine and plasma was measured with a vapor-pressure osmometer (Osmomat 030, Gonotec, Berlin, Germany).
Membrane Fractionation for Immunoblotting
After rapid removal of kidneys, the left kidney was frozen in liquid nitrogen and the right kidney was split into inner medulla (IM), inner stripe of outer medulla (ISOM), and cortex and outer stripe of outer medulla (C+OSOM) using a microscope. Tissue (IM, ISOM, or C+OSOM) was minced finely and homogenized in 1 (IM), 1.5 (ISOM), or 2 ml (C+OSOM) of ice-cold dissecting buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2, and containing the following protease inhibitors: 8.5 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride), with five strokes of a motor-driven Potter-Elvehjem homogenizer at 1,250 rpm. This homogenate was centrifuged in a Universal 30RF centrifuge (Hettich, Tuttlingen, Germany) at 4,000 g for 15 min at 4°C. The supernatants were assayed for protein concentration using the BCA protein assay method. Gel samples (in Laemmli sample buffer containing 2% SDS) were made from this membrane preparation and then stored at –20°C.
Electrophoresis and Immunoblotting
Samples of membrane fractions from IM, ISOM, and C+OSOM were run on 12 or 9% SDS-polyacrylamide minigels (Bio-Rad Mini Protean III). For each gel, an identical gel was run in parallel and subjected to Coomassie blue staining. The Coomassie-stained gel was used to verify identical loading or to allow for potential correction for minor differences in loading after scanning and densitometry of major bands. The other gel was subjected to Western blot analysis. After transfer by electroelution to nitrocellulose membranes, blots were blocked with 5% milk in PBS-T (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h and incubated with primary antibodies overnight at 4°C. After being washed with PBS-T, the blots were incubated with horseradish peroxidase-conjugated secondary antibody (diluted 1:3,000, P448, DAKO, Glostrup, Denmark) for 1 h. After a final washing as above, antibody binding was visualized using the enhanced chemiluminescence system (Amersham International). Enhanced chemiluminescence films with bands within the linear range were scanned (Arcus II, Agfa, and Corel Photopaint software). The labeling density was corrected by densitometry of the Coomassie blue-stained gels.
Immunocytochemistry
The kidneys from BUO rats and sham-operated rats were fixed by retrograde perfusion via the abdominal aorta with 3% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4. For immunoperoxidase microscopy, kidney blocks containing all kidney zones were dehydrated and embedded in paraffin. The paraffin-embedded tissues were cut at 2 µm on a rotary microtome (Leica, Heidelberg, Germany). The sections were deparaffinated and rehydrated. For immunoperoxidase labeling, endogenous peroxidase was blocked by 0.5% H2O2 in absolute methanol for 10 min at room temperature. To reveal antigens, sections were put in 1 mmol/l Tris solution (pH 9.0) supplemented with 0.5 mM EGTA and heated for 10 min using a microwave oven. Nonspecific binding of immunoglobulin was prevented by incubating the sections in 50 mM NH4Cl in 30 min followed by blocking in PBS supplemented with 1% BSA, 0.05% saponin, and 0.2% gelatin. Sections were incubated overnight at 4°C with primary antibodies diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100. After a rinse with PBS supplemented with 0.1% BSA, 0.05% saponin, and 0.2% gelatin for 3 x 10 min, the sections were washed, then incubated with horseradish peroxidase-conjugated immunoglobulin (1:200, P448, DAKO) diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100. The sections were washed for 3 x 10 min, followed by incubation with diaminobenzidine for 10 min. The microscopy was carried out using a Leica DMRE light microscope (Leica).
Primary Antibodies
For semiquantitative immunoblotting and immunocytochemistry, we used previously characterized polyclonal antibodies to several key acid-base transporters, as follows: NHE3 (26); NBC1 (23); NBCn1 (23); NKCC2 (BSC-1) (9); pendrin (10); and H+-ATPase, B1 subunit of vacuolar type H+-ATPase (20).
Statistical Analysis
For the densitometry of immunoblots, samples from experimental kidneys were run on each gel with corresponding sham kidneys. Renal acid-base transporter expression in the samples from the experimental animals was calculated as a fraction of the mean sham control value for that gel. Parallel Coomassie blue-stained gels were subjected to densitometry and used for correction of potential minor differences in loading. Values are presented as means ± SE. Comparisons between two groups were made by unpaired t-test, those among more than two groups were made by one way ANOVA. P values <0.05 were considered statistically significant.
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RESULTS |
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As shown in Table 1, plasma pH and levels of HCO3– and total CO2 in rats with 24-h BUO (protocol 1) were significantly lower than that in sham-operated rats. Moreover, actual and standard base excess and standard bicarbonate concentration were also markedly decreased (Table 1) in BUO rats, indicating that BUO was associated with severe metabolic acidosis.
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Four days after release of the obstruction (protocol 2, Table 2), plasma pH did not differ between BUO-R rats and controls, whereas the plasma levels of HCO3– and total CO2 were slightly increased in BUO-R rats compared with Sham. To examine whether release of BUO was associated with a urinary acidification defect, rats were given NH4Cl for 2 days. Plasma pH and plasma HCO3– levels were significantly reduced in rats with BUO-A compared with both Sham-A rats and BUO-R rats without acid loading (Table 2), which indirectly indicates reduced urinary acid excretion after release of the obstruction, consistent with the results of previous studies (6, 22, 36, 37, 41).
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In rats with BUO, protein abundance of NHE3 was significantly decreased in C+OSOM to 21% of the level in sham-operated rats (Table 3 and Fig. 2, A and B). After release of the obstruction, the observed reduction of NHE3 was partially reversed to 64% of the level in sham-operated rats (Table 4). Semiquantitative immunoblotting also revealed a marked reduction of NBC1 expression levels in BUO rats compared with Sham (Table 3 and Fig. 3, A and B). This finding was confirmed by immunocytochemistry. As seen in Fig. 3, NBC1 was located in the basolateral plasma membrane of the proximal tubules in both BUO rats (Fig. 3, C and E) and sham-operated control rats (Fig. 3, D and F). Consistent with immunoblotting, the labeling density of NBC1 was weaker in BUO rats than Sham. Unlike NHE3, semiquantitative immunoblotting revealed that the expression levels of NBC1 completely recovered after release of the obstruction (94 ± 6 vs. 100 ± 4%, P > 0.05, Table 4).
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Semiquantitative immunoblotting revealed that NHE3 expression in the TAL was significantly decreased in rats with 24-h BUO compared with Sham rats (Table 3 and Fig. 2, C and D) and NHE3 expression levels remained low 4 days after release of BUO (64 ± 15 vs. 100 ± 7%, P < 0.05, Table 4). The abundance of NBCn1, which is localized in the basolateral plasma membrane of TAL cells in the outer medulla (7, 39), was significantly decreased in BUO (Table 3 and Fig. 4, A and B) and after release of BUO (Table 4). Immunocytochemistry revealed NBCn1 labeling at the basolateral plasma membrane of TAL cells and intercalated cells of CD in the ISOM of the kidneys from both BUO rats (Fig. 4, C and E) and Sham control rats (Fig. 4, D and F). The labeling intensity was markedly decreased in BUO rats. Semiquantitative immunoblotting demonstrated that the abundance of NKCC2 in ISOM was markedly reduced in BUO to 3% of Sham (Table 3 and Fig. 2, E and F). The abundance of NKCC2 was partially reversed after release of BUO (64 ± 15 vs. 100 ± 9%, P < 0.05, Table 4).
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The CD is an important segment for acid-base regulation. Semiquantitative immunoblotting demonstrated that the abundance of H+-ATPase was slightly increased in the IM of kidneys from BUO rats (Table 3 and Fig. 5, A and B) compared with Sham; however, the abundance of H+-ATPase in the C+OSOM and ISOM was unchanged in BUO rats (Table 3 and Fig. 5, C–F). This finding was confirmed by immunocytochemistry. As seen in Fig. 5, anti-H+-ATPase antibody labeled the apical plasma membrane domains of intercalated cells of connecting tubule and cortical CD. The labeling intensity did not differ between BUO (Fig. 5, G and I) and Sham rats (Fig. 5, H and J). In contrast to the findings in BUO rats, protein expression levels of the H+-ATPase was markedly decreased in both the ISOM (57 ± 10 vs. 100 ± 7%, P < 0.05, Table 4) and IM (19 ± 3 vs. 100 ± 12%, P < 0.05, Table 4) 4 days after release of BUO.
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The Na+-independent Cl–/HCO3–/OH– exchanger pendrin is expressed in the apical regions of type B and non-A, non-B intercalated cells within the cortical CD, the connecting tubule, and the distal convoluted tubule, where it mediates HCO3– secretion and Cl– absorption (21, 34, 40). Semiquantitative immunoblotting demonstrated that the abundance of pendrin in the C+OSOM was significantly reduced in BUO rats compared with sham-operated controls (Table 3 and Fig. 6, A and B). Consistent with this, immunohistochemical analysis demonstrated a mild reduction in the labeling intensity of BUO kidneys with no major change in the subcellular localization of pendrin in intercalated cells within the cortex of BUO rats (Fig. 6, C and E) compared with sham controls (Fig. 6, D and F). After release of BUO, pendrin expression recovered to control levels (100 ± 4 vs. 100 ± 5%, P > 0.05, table 4).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Metabolic Acidosis in BUO and Urinary Acidification Defect after Release of BUO
The metabolic acidosis seen in BUO rats could be due to reduced renal blood flow, reduced glomerular filtration rate (11), and, consequently, accumulation of acidic metabolic waste in the body. Moreover, reduced renal HCO3– reabsorption mediated by Na+/HCO3– cotransporters may at least partially contribute to development of acidosis.
To examine urinary acidification ability after release of BUO, BUO-R rats were subjected to acid loading with NH4Cl, demonstrating that plasma pH and HCO3– were much lower in BUO-A rats compared with Sham-A rats, which maintained normal plasma pH and HCO3– level in response to acid loading. This finding indicates development of a urinary acidification defect after release of the obstruction, as demonstrated previously (2, 3, 15, 37, 38, 41). Although measurements of urinary excretion of HCO3– and titratable acid would provide a more complete result, the present finding is consistent with previous studies demonstrating impaired urinary acidification in the kidney after obstruction (2, 3, 15, 37, 38, 41).
Reduced Expression of NBC1 and NHE3 in the Proximal Tubule of Rats with BUO and Release of BUO
Functional studies (4, 15) and immunohistochemical analyses (20, 27) have revealed that NBC1 is expressed in the basolateral plasma membrane of rat proximal tubules, predominantly in S1 and S2 segments, suggesting a role of NBC1 in mediating electrogenic HCO3– efflux to the blood circulation (4) . Human patients with proximal renal tubular acidosis resulting from mutations in NBC1 have been reported (8, 16, 17), and NBC1–/– mice have also been reported, to exhibit severe metabolic acidosis (13), thereby confirming a HCO3–-absorptive role for NBC1 in the kidney. The present study revealed that NBC1 expression was significantly decreased in the kidney cortex of rats with BUO, suggesting reduced NBC1-mediated HCO3– reabsorption in the proximal tubule. It is well established that apical NHE3 is involved in large quantities of transcellular HCO3– reabsorption (33, 35, 42) in conjunction with basolateral NBC1 in the proximal tubules. Consistent with our previous study (26), NHE3 abundance was dramatically reduced in response to BUO and release of BUO. HCO3– wasting resulting from reduced NHE3-mediated H+ secretion and reduced NBC1-mediated HCO3– reabsorption in BUO will unavoidably result in development and maintenance of proximal tubular acidosis and development of a urinary acidification defect, as observed in rats after release of BUO. After release of BUO, both NHE3 and NBC1 abundance recovered by different degrees, accompanied by an improved plasma HCO3– level in rats with BUO-R. However, how ureteral obstruction downregulates renal NBC1 expression in proximal tubule epithelial cells has not been explained in the present study, and further studies are needed to identify the signaling pathways involved in this.
Reduced Expression of NBCn1 and NKCC2 in the TAL of Rats with BUO and Release of BUO
We demonstrated that the expression of NBCn1 was significantly decreased in the TAL of rats with BUO and release of BUO. NBCn1 localizes in the basolateral plasma membrane of TAL cells in the OM (7, 39), plays an important role in transporting HCO3– into cells, maintaining intracellular pH levels, and facilitating ammonium reabsorption (23, 30) by importing HCO3– to the cells for buffering protons formed by the dissociation of NH4+ (NH3 and H+) that is taken up across the luminal membrane. It is suggested that absorption of NH4+ in the medullary TAL is mediated by a coordinated process of the NKCC2 (presumably by substituting for K+) and basolateral NBCn1 (23, 30). Thus reduced basolateral NBCn1 expression in rats with BUO is likely involved in the urinary acidification defect. Downregulation of NBCn1 may also be considered as an adaptation to decreased apical NKCC2 expression, consistent with previous studies (26). Potentially, the reduction of NKCC2 abundance in BUO rats results in decreased NH4+ absorption in the mTAL and NH3 accumulation in the medulla, thus affecting H+ excretion in urine after release of BUO, which may further aggravate the urinary acidification defect.
Altered Expression of H+-ATPase in CD of Rats with BUO and Release of BUO
The CD is the final and most important segment in the kidney to maintain homeostasis by acidification of urine (33) and plays an important role in the development of impaired urine acidification in response to obstruction, as previously demonstrated (32). In the present study, we demonstrated that H+-ATPase abundance in rats with 24-h BUO was unchanged in the C+OM, but it was significantly increased in the IM. This finding may be a compensatory result of the inability of the obstructed kidney to excrete catabolic acidic metabolites. Interestingly, however, release of BUO was associated with markedly decreased expression of H+-ATPase in the medullary CD, supporting the view that a distal renal tubular acidosis may occur early after release of the obstruction. The postobstructive acidification defects may be caused by both decreased protein abundance and enzyme activity. Consistent with this, Valles et al. (38) demonstrated that the activity of H+-ATPase was reduced in the medullary CD, causing a defect in urinary H+ excretion in rats with unilateral ureter obstruction, and Purcell et al. (31) showed that changes in the cellular distribution of H+-ATPase occur in intercalated cells throughout the CD after obstruction.
Pendrin is localized in the apical membrane in type B cells in the CD and functions as secreting HCO3– for exchange of Cl– (15, 21, 34, 40). A previous study in pendrin-deficient mice revealed an essential role of pendrin in renal HCO3– secretion in the cortical CD (34). The present study demonstrated a reduction in pendrin expression in the cortical CD in rats with BUO with no major change in the subcellular localization of pendrin. Potentially, the reduced pendrin expression may represent a compensatory phenomenon to correct for the BUO-associated acidosis by reducing tubular HCO3– secretion.
In conclusion, the present study demonstrated marked changes in the protein expression of a number of membrane proteins important for renal acid-base regulation during and after release of urinary tract obstruction, coinciding with an impairment of urinary acid excretion. In particular, reduced NHE3 and NBC1 expression in the proximal tubules and reduced H+-ATPase expression in the CD may contribute to renal tubular acidosis and the urinary acidification defect associated with BUO and release of BUO. Reduced expression of NBCn1 in the TAL and pendrin in the CD may represent compensatory changes to the acidosis.
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ACKNOWLEDGMENTS |
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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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87-kDa band. B and D: densitometric analysis (corrected according to densitometry of Commassie blue-stained gels and loading fraction of C+OSOM and ISOM) of all samples from BUO and sham-operated controls revealed a significant decrease in NHE3 levels to 21 ± 4% in the C+OSOM and to 27 ± 2% in the ISOM in obstructed kidneys (*P < 0.05). E: immunoblots were reacted with affinity-purified type 1 bumetanide-sensitive Na+-K+-2Cl– cotransporter (NKCC2) antibody and revealed a strong, broad band of 146- to 176-kDa molecular mass centered at 
