AJP - Renal Add DOIs to your references at manuscript stage!
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Renal Physiol 293: F416-F427, 2007. First published April 4, 2007; doi:10.1152/ajprenal.00391.2006
0363-6127/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/1/F416    most recent
00391.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mohebbi, N.
Right arrow Articles by Wagner, C. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mohebbi, N.
Right arrow Articles by Wagner, C. A.

Thyroid hormone deficiency alters expression of acid-base transporters in rat kidney

Nilufar Mohebbi,1,2 Jana Kovacikova,1 Marta Nowik,1 and Carsten A. Wagner1

1Institute of Physiology and Zurich Center for Human Integrative Physiology, University of Zurich, Zurich, Switzerland; and 2Innere Klinik, HELIOS Klinikum Berlin, Medical Faculty of the Charité, Humboldt University of Berlin, Berlin, Germany

Submitted 3 October 2006 ; accepted in final form 3 April 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hypothyroidism in humans is associated with incomplete distal renal tubular acidosis, presenting as the inability to respond appropriately to an acid challenge by excreting less acid. Here, we induced hypothyroidism in rats with methimazole (HYPO) and in one group substituted with L-thyroxine (EU). After 4 wk, acid-base status was similar in both groups. However, after 24 h acid loading with NH4Cl HYPO rats displayed a more pronounced metabolic acidosis. The expression of the Na+/H+ exchanger NHE3, the Na+-phosphate cotransporter NaPi-IIa, and the B2 subunit of the vacuolar H+-ATPase was reduced in the brush-border membrane of the proximal tubule of the HYPO group, paralleled by a lower abundance of the Na+/HCO3– cotransporter NBCe1 and a higher expression of the acid-secretory type A intercalated cell-specific Cl–/HCO3– exchanger AE1. In contrast to control conditions, the expression of NBCe1 was increased in the HYPO group during metabolic acidosis. In addition, net acid excretion was similar in both groups. The relative number of type A intercalated cells was increased in the connecting tubule and cortical collecting duct of the HYPO group during acidosis. Thus thyroid hormones modulate the renal response to an acid challenge and alter the expression of several key acid-base transporters. Mild hypothyroidism is associated only with a very mild defect in renal acid handling, which appears to be mainly located in the proximal tubule and is compensated by the distal nephron.

collecting duct; intercalated cells; metabolic acidosis; hypothyroidism

THE KIDNEY PLAYS A CENTRAL role in the regulation and maintenance of acid-base homeostasis by reabsorbing bicarbonate, excretion of acids, and regeneration of bicarbonate both in the proximal tubule during ammoniagenesis and in the collecting duct. The importance of these mechanisms has been highlighted by a number of inherited or acquired disorders in humans leading to syndromes of incomplete or complete renal tubular acidosis (14, 19). Renal acid-base transport is tightly regulated by several factors including various hormones and closely linked to daily metabolism to ensure acid-base equilibrium.

Thyroid hormones regulate basic metabolism in many organs and cells and are critical for normal development as well as normal cellular function. Also, kidney development and function are influenced by thyroid hormones. Both a complete absence of thyroid hormones and hypothyroidism are frequently associated with abnormal kidney development and/or impaired kidney function.

A number of important renal transport proteins are decreased in their expression and function in the absence of thyroid hormones as demonstrated for the Na+-phosphate cotransporter NaPi-IIa in rat kidney (2, 35) and the renal opossum kidney cell line (2) or the Na+-sulfate cotransporter NaSi in rat kidney (34). Also, Na+/Ca2+ exchanger activity in the brush-border membrane (BBM) was reduced (23), Na+-K+-ATPase activity decreased (11), and the expression of the thick ascending limb of Henle's loop-specific, loop-diuretic sensitive Na+-K+-2Cl– cotransporter (NKCC2) was lower (11). The abundance of the vasopressin-regulated aquaporin-2 (AQP2) water channel in the collecting duct has been reported to be either reduced or increased during hypothyroidism (11, 35).

In addition to the effects on expression of these renal transport proteins, thyroid hormones seem to also affect the expression and/or function of renal acid-base transporters. In thyroidectomized rats, the absence of thyroid hormones leads to reduced expression of the major Na+/H+ exchanger, NHE3, during maturation of the proximal tubule (4, 18) and also in adult kidney (11, 35). Functionally, hypothyroidism is associated with impaired renal bicarbonate reabsorption after bicarbonate loading (28), reduced H+ secretion in the distal nephron (29), a decreased urinary-blood PCO2 gradient typical of distal renal tubular acidosis (27), and the impaired ability to acidify urine and excrete ammonium after an acute NH4Cl load (26). Furthermore, incomplete distal renal tubular acidosis is frequently observed in patients with hypothyroidism without other signs of renal disease (31). Induction of metabolic acidosis in healthy humans reduces serum-free thyroxine (T4; fT4) levels and increases thyroid-stimulating hormone (TSH) concentrations (10).

Thus thyroid hormone appears to positively modulate renal expression and function of acid-base transporters and the ability to maintain acid-base homeostasis. However, with the exception of the Na+/H+ exchanger NHE3, it has remained unknown which acid-base transport proteins are modulated by thyroid hormones. Therefore, we used a pharmacological model of mild hypothyroidism in rats and investigated acid-base status, renal acid excretion, and expression of acid-base transporters under basal conditions and during a short-term acid load. Despite lower expression of several acid-base transporters, renal acid excretion was almost normal in hypothyroid rats. Nevertheless, hypothyroid rats developed more severe metabolic acidosis possibly due to the lack of other compensatory mechanisms.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal Model

Male Wistar rats (Harlan Teklad), weighing 250–300 g, were divided equally into two groups: hypothyroid (HYPO) and hypothyroid with L-thyroxine replacement (EU). Twelve rats in each study group were used for immunoblotting and metabolic cage studies, and 10 in each group were used for immunofluorescence studies. Both groups were maintained on the same standard diet and had free access to drinking water. Body weights were documented weekly.

Hypothyroidism was induced pharmacologically as described previously (32, 35) by adding 0.05% methimazole to the drinking water for 4 wk and was confirmed by measurement of serum-free thyroxine and TSH levels in blood serum. EU rats received the same methimazole treatment but were also substituted with L-thyroxine (2 µg·100 g body wt–1·day–1) in the drinking water. After 4 wk of treatment, animals designated for metabolic cage studies were placed into individual cages. One-half of the rats in each group was then treated with either 2% sucrose/0.28 M NH4Cl or 2% sucrose alone in the drinking water for 24 and 36 h to induce mild metabolic acidosis.

Parameters of systemic acid-base status and electrolytes were measured immediately in arterial blood from anesthetized rats of both groups under control conditions and after oral loading with 0.28 M NH4Cl for 24 and 36 h on a Radiometer ABL 505 (Radiometer, Copenhagen, Denmark) blood-gas analyzer. Urine analysis was performed on urine collected under mineral oil.

All animal experiments were performed according to national and international guidelines and laws of animal welfare and protocols approved by the local Veterinary Authority (Veterinäramt Zurich) under protocol number 70/2006.

Blood and Urine Analysis

TSH and fT4 were measured by ELISA in plasma samples collected as described above (Vet Med Labor, Division of IDEXX Laboratories, Ludwigsburg, Germany). Electrolytes in blood were measured on a Radiometer ABL 505 blood-gas analyzer. Ion chromatography (Metrohm ion chromatograph) was performed to obtain the following values in urine samples: K+, Na+, Cl–, Ca2+, Mg2+. Serum creatinine concentration was measured using a kit based on the F-Daos method (Wako Chemicals), and urine creatinine was analyzed applying the Jaffé method (36, 37). Urine ammonium concentration was determined according to the Berthelot protocol (6). Determination of titratable acids was done as described previously (12). Briefly, a mixture of equal volumes of urine and 0.1 N HCl was boiled for 2 min. After the mixture was cooled for 10 min, titration with 0.1 N NaOH to pH 7.40 at 37°C was performed. A blank sample (distilled water) was treated in an identical way. The difference in volumes of NaOH required to titrate the sample and the blank, multiplied by the normality of NaOH, times 1,000, revealed the concentration of titratable acids (in mmol/l). Osmolarity of blood and urine was determined using a Roebling osmometer (Auer Bittmann Sourié) by examination of freezing-point depression. Glomerular filtration rates, fractional excretion of ions, anion gap in blood and urine, and net acid excretion were calculated from the measured values.

Immunoblotting

Rats were anesthetized with ketamine-xylazine (ip) perfused through the left ventricle with warm (37°C) sucrose/phosphate buffer (140 mM sucrose, 28 mM NaH2PO4, 112 mM Na2HPO4, pH 7.4), and the kidneys were removed rapidly and frozen until further analysis. Frozen kidneys were then used for crude membrane and BBM preparation as described previously using the Mg2+-precipitation technique (7). After measurement of the protein concentration (Bio-Rad Protein kit), 50 µg of crude membrane protein or 20 µg of BBM protein was solubilized in Laemmli sample buffer, and SDS-PAGE was performed on 8–12% polyacrylamide gels. Proteins were transferred electrophoretically from gels to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Bedford, MA). After blocking with 5% milk powder in Tris-buffered saline containing 0.1% Tween 20 for 60 min, the blots were incubated with the respective primary antibodies overnight at 4°C or 2 h at room temperature. After washing and subsequent blocking, blots were incubated with the secondary antibody for 1 h at room temperature. Antibody binding was detected with an enhanced chemiluminescence ECL kit (Amersham Pharmacia Biotech) or with CDP-Star (Roche Diagnostics, Indianapolis, IN), respectively, using the DIANA III-chemiluminescence detection system (Raytest, Straubenhardt, Germany). All images were analyzed using appropriate software (Advanced Image Data Analyzer, Raytest) to calculate the protein of interest/actin ratio.

Immunohistochemistry

Rats were perfused through the left ventricle with PBS followed by a paraformaldehyde-lysine-periodate fixative. Kidneys were removed and fixed overnight at 4°C by immersion in paraformaldehyde-lysine-periodate. Kidneys were washed three times with PBS, and after cryoprotection with PBS/30% sucrose solution for at least 12 h, thin coronal slices were cut, mounted on thin cork plates, and frozen in liquid propane cooled with liquid nitrogen. Immunostaining was carried out as described previously (39). Sections were incubated with 1% SDS for 5 min, washed three times with PBS, and incubated with PBS containing 1% BSA for 15 min before application of the primary antibody. The primary antibodies (see below) were diluted in PBS and applied either for 75 min at room temperature or overnight at 4°C. Sections were then washed twice for 5 min with high-NaCl-PBS (PBS+2.7% NaCl), once with PBS, and incubated with dilutions of the secondary antibodies (see below) at the given dilutions for 1 h at room temperature. Sections were again washed twice with high-NaCl-PBS and once with PBS before being mounted with Vectashield (Vector Laboratories, Burlington, CA). Sections were viewed using a LCSM Leica confocal microscope. Pictures were processed (overlaid) using Adobe Photoshop software.

Cell counting for the different subtypes of intercalated cells and the subcellular localization of the B1 subunit of the vacuolar H+-ATPase was performed as described previously (3, 8, 39).

Antibodies

The following primary antibodies were used in this study at the given dilutions.

Primary antibodies were detected for immunoblotting using secondary antibodies coupled to either alkaline phosphatase (1:5,000, Promega) or horseradish peroxidase (1:10,000, Amersham Biosciences) and using the appropriate detection systems (see above). For indirect immunohistochemistry, donkey anti-guinea pig Cy3 (1:1,000, Jackson ImmunoResearch Laboratories), donkey anti-mouse Alexa 488 (1:400, Molecular Probes), and donkey anti-goat Alexa 488 (1:400, Molecular Probes) were used as secondary antibodies.

Statistical Analysis

Results are expressed as means ± SE. All data were tested for significance using ANOVA and an unpaired Student's t-test, and only results with P < 0.05 were considered as significant.Go


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Animal Model

Animals treated with methimazole alone for 4 wk had mild hypothyroidism (HYPO group)as determined by serum analysis for TSH and fT4 levels (Table 1). fT4 levels were reduced ~10-fold, and TSH levels increased similar to what has been described in this and similar models of hypothyroidism (11, 35, 41). A second group of animals was treated with methimazole but with L-thyroxine substituted and showed normal fT4 and TSH levels as described in various rat models (15, 35). This group served as the euthyroid control group (EU).


View this table:
[in this window]
[in a new window]

  		Table 1. Blood and urine parameters from euthyroid and hypothyroid rats

 
Mild hypothyroidism was associated with a lower growth rate in the HYPO group as evident from a lower body weight after a 4-wk treatment (Table 1). Blood analysis showed no difference in arterial blood gases or acid-base status between the EU and HYPO groups (Table 1). However, serum phosphate concentration was significantly lower in the HYPO group.

Similarly, urinary parameters showed no significant changes in pH and osmolality. However, compared with the EU group HYPO rats excreted significantly more volume, phosphate, and chloride in urine but had a similar rate of net acid excretion (Table 1).

To test for renal acid excretion and adaption to an acid challenge, rats were given NH4Cl in the drinking water for 24 h, and blood and urine values were monitored (Table 2). Induction of metabolic acidosis decreased TSH levels in contrast to observations in healthy human volunteers with chronic metabolic acidosis (10). NH4Cl loading for 24 h induced in the EU rats a lower arterial HCO3– level, an increase in serum chloride concentrations, an appropriate fall in arterial PCO2, and no change in arterial pH, consistent with a mild hyperchloremic metabolic acidosis with respiratory compensation (Table 2). In contrast, in the HYPO group, a much more pronounced metabolic acidosis was observed with a stronger decrease in HCO3–, a lower arterial pH, higher chloride concentration, and a lower PCO2, indicating severe metabolic acidosis with an inappropriately low respiratory compensation (Table 2). After 36 h, the EU group also developed metabolic acidosis, but arterial HCO3– levels remained higher and PCO2 returned to almost normal levels, indicating that other mechanisms contributed to the adaption and compensation whereas in the HYPO group this adaptive response seemed to be less effective (Fig. 1). Thus mild hypothyroidism impairs normal adaption to an oral acid load.


View this table:
[in this window]
[in a new window]

  		Table 2. Blood and urine parameters from euthyroid and hypothyroid rats following 24 h of NH4Cl loading

 

Figure 1
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 1. Arterial blood-gas values and urinary acid excretion. Shown is a summary of blood-gas values of euthyroid (EU; methimazole+L-thyroxine) and hypothyroid (HYPO; methimazole) rats under control conditions and after 24 or 36 h of oral acid loading (0.28 M NH4Cl). A–C: no differences were observed in arterial blood pH, bicarbonate levels, and PCO2 under control conditions. However, after 24 h acid-loaded hypothyroid rats displayed a severe metabolic acidosis with lower pH and bicarbonate levels and a compensatory fall in PCO2. Also, euthyroid rats had lower bicarbonate levels but they remained higher than in the hypothyroid group. After 36 h of acid loading, both groups were acidotic; however, euthyroid rats compensated better. D: time course of net acid excretion of euthyroid and hypothyroid rats after oral acid loading. Urine was collected over several periods and analyzed for net acid excretion (NAE). No significant difference was found between both groups; n = 10 each group *Significant difference between euthyroid and hypothyroid rats, P < 0.05.

 
Urine analysis revealed that rats in both groups acidified their urine to a similar extent (Table 2). Also, excretions of ammonium and total net acid were almost equal in both groups. However, HYPO rats excreted more phosphate, which may assist in excreting protons bound to titratable acids (Tables 1 and 2). No significant bicarbonaturia was found in HYPO rats. A more detailed analysis of net acid excretion over the 36 h confirmed similar rates of urinary acid elimination. HYPO rats, however, showed a tendency for reduced creatinine clearance, indicating a lower glomerular filtration rate. Hence, mild hypothyroidism did not significantly impair renal excretion of an acute oral acid load.

Altered Abundance of Renal Acid-Base Transport Proteins in Mild Hypothyroidism

Proximal tubular transport proteins. Immunoblotting showed a marked decrease in the protein abundance of the major renal Na+/H+exchanger NHE3 and the Na+-phosphate cotransporter NaPi-IIa in the brush-border membrane of HYPO rats compared with EU rats as described previously (Fig. 2). Similarly, the abundance of the B2 subunit of the vacuolar H+-ATPase was slightly reduced in the BBM of HYPO rats. The a4 subunit, however, showed the same abundance in the BBM of EU and HYPO rats. In agreement with the lower abundance of transport proteins involved in apical bicarbonate reabsorptive steps, the expression level of the basolateral electrogenic Na+-bicarbonate cotransporter, NBCe1, providing the exit step was also reduced.


Figure 2
View larger version (39K):
[in this window]
[in a new window]

  		Fig. 2. Expression of proximal tubular acid-base-transporting proteins. A: immunoblotting of Na+/H+ exchanger NHE3, the B2 (ATP6V1B2) and a4 (ATP6V0A4) H+-ATPase subunits, and the Na+-phosphate cotransporter NaPi-IIa in brush-border membranes from euthyroid (methimazole+L-thyroxine) and hypothyroid rats (methimazole). The abundance of the basolateral electrogenic bicarbonate transporter NBCe1 was tested in total membranes. All immunoblot membranes were stripped and reprobed for actin. B: bar graphs summarizing data from immunoblotting. All data were normalized against actin. The abundance of NHE3, the B2 subunit, NaPi-IIa, and NBCe1 was decreased. *Significant difference between euthyroid and hypothyroid rats, P < 0.05.

 
Distal nephron: AE1 and pendrin. Hypothyroidism was associated with a significant increase in the total kidney protein abundance of the type A intercalated cell-specific Cl–/HCO3– exchanger AE1. In contrast, the non-type A intercalated cell-specific Cl–/HCO3– exchanger pendrin was not altered despite higher urinary chloride excretion. Also, the expression of the a4 subunit (ATP6V0A4) of the vacuolar H+-ATPase was not affected in a total membrane preparation of whole kidney (Fig. 3).


Figure 3
View larger version (41K):
[in this window]
[in a new window]

  		Fig. 3. Expression of acid-base transporting proteins in the distal nephron. A: abundance of AE1, pendrin, and the a4 H+-ATPase subunit was tested by immunoblotting. All membranes were reprobed for actin. B: normalized data demonstrate increased AE1 expression in hypothyroid rats whereas pendrin and a4 showed no difference. *Significant difference between euthyroid and hypothyroid rats, P < 0.05.

 
Acid-Base Transport Proteins After Induction of Metabolic Acidosis

Proximal tubular proteins. Similar to control conditions, immunoblotting of protein abundance of NaPi-IIa showed a marked reduction after 24 h of metabolic acidosis in HYPO rats vs. EU animals, consistent with a higher rate of urinary phosphate excretion. Despite a more severe metabolic acidosis in the HYPO group after 24-h acid loading, the abundance of the apical proton-extruding proteins remained lower in the HYPO group as shown for NHE3 and the B2 subunit of the vacuolar H+-ATPase. The expression levels of the a4 subunit were not different between HYPO and EU rats. In contrast, the basolateral NBCe1 transporter was enhanced in its expression in the HYPO group (Fig. 4).


Figure 4
View larger version (44K):
[in this window]
[in a new window]

  		Fig. 4. Expression of proximal tubular acid-base-transporting proteins after acid loading. A: immunoblotting of NHE3, the B2 (ATP6V1B2) and a4 (ATP6V0A4) H+-ATPase subunits, and the Na+-phosphate cotransporter NaPi-IIa in brush-border membranes, and NBCe1 in total-membrane preparation after 24 h of acid loading. All immunoblot membranes were stripped and reprobed for actin. B: bar graphs summarizing data from immunoblotting. All data were normalized against actin. The abundance of NHE3, the B2 subunit, NaPi-IIa, was lower in hypothyroid rats, whereas the expression of NBCe1 increased. *Significant difference between euthyroid and hypothyroid rats, P < 0.05.

 
Distal nephron: AE1 and pendrin. Similar to normal conditions, the abundance of the Cl–/HCO3– exchanger AE1 was higher in the HYPO group after 24 h of NH4Cl loading (Fig. 5). Expression levels of pendrin and the a4 H+-ATPase subunit remained similar in both groups. We also analyzed the subcellular localization of the B1 proton pump subunit as an indicator of proton pump trafficking in response to the different metabolic status of the animals. As summarized in Table 3 and shown in Fig. 6, there was a shift of staining for the proton pumps from the apical membrane domain to a more diffuse cytosolic staining in the cortical collecting duct of HYPO rats, consistent with a possible reduction in the ability to secrete protons. However, after NH4Cl loading, H+-ATPase staining was prominent at the apical membrane in EU and HYPO rats, and no difference could be detected between both groups.


Figure 5
View larger version (37K):
[in this window]
[in a new window]

  		Fig. 5. Expression of acid-base-transporting proteins in the distal nephron after acid loading. A: abundance of AE1, pendrin, and the a4 H+-ATPase subunit was assessed after 24 h of acid loading. All membranes were reprobed for actin. B: normalized data demonstrate increased AE1 expression in hypothyroid rats during acidosis, whereas pendrin and a4 remained unaltered. *Significant difference between euthyroid and hypothyroid rats, P < 0.05.

 

View this table:
[in this window]
[in a new window]

  		Table 3. Subcellular distribution of H+-ATPases in intercalated cells in kidneys from euthyroid and hypothyroid rats

 

Figure 6
View larger version (35K):
[in this window]
[in a new window]

  		Fig. 6. Remodeling of the connecting tubule (CNT) and CCD. Kidneys were stained with antibodies against pendrin or AE1 (red) and calbindin D28k/AQP2 (combined together and shown in green) as markers of non-type A intercalated cells and principal cells, respectively. A and B: staining for pendrin in the CNT of euthyroid and hypothyroid rats after 48 h of acidosis. Non-type A intercalated cells (stained in red with pendrin) are indicated by an asterisk and type A intercalated cells (not stained for pendrin or calbindin D28k/AQP2) by arrowheads. C and D: AE1 in type A intercalated cells (arrowheads) in the outer medullary collecting duct (OMCD) of euthyroid and hypothyroid rats after 36 h of acidosis. E–H: segment-specific analysis of relative cell numbers demonstrated no changes in the CNT under control conditions but a marked increase of type A intercalated cells after 36 h of acidosis in the hypothyroid group. Non-type A intercalated cells were reduced during acidosis, whereas principal cells showed also a small increase in hypothyroid rats. Similarly, in the CCD of hypothyroid rats a small decrease in type A intercalated cells was seen under control conditions, whereas the relative number of type A intercalated cells strongly increased after 48 h of acidosis. The relative number of non-type A intercalated cells was greatly reduced. *Significant difference between euthyroid and hypothyroid rats, P < 0.05.

 
Remodeling of the Distal Nephron

Immunohistochemistry was performed to test how hypothyroidism affected the relative abundance of acid-secretory type A intercalated cells and bicarbonate-secretory non-type A intercalated cells. We used AE1 and pendrin as specific markers of type A intercalated cells and non-type A intercalated cells, respectively. Principal cells were marked with calbindin D28k and the AQP2 water channel (24). Detailed analysis of the different segments of the collecting system showed no apparent difference in the subcellular localization of pendrin or AE1 between the EU and HYPO groups under control conditions and during acidosis (not shown). Also, the relative number of type A and non-type A intercalated cells as well as principal cells in the CNT was similar in the EU and HYPO groups under control conditions (Table 4, Fig. 7). A small decrease in type A intercalated cells in the cortical collecting duct of HYPO rats was noted. In contrast, during metabolic acidosis a marked increase in type A intercalated cells in both the connecting segment and cortical collecting duct at the expense of non-type A intercalated cells was noted (Table 4, Fig. 7). However, in the outer medullary collecting duct and the initial inner medullary collecting duct no difference in the relative abundance of type A intercalated cells and principal cells could be observed (Table 4). We did not observe any cells positive for markers of both subtypes of intercalated cells or stained for markers of principal and intercalated cells. Thus mild hypothyroidism and metabolic acidosis led to a remodeling of the initial parts of the collecting system with an increase in the relative number of acid-secretory type A intercalated cells.


View this table:
[in this window]
[in a new window]

  		Table 4. Relative abundance of type A and B intercalated cells and principal cells, respectively, in kidneys from euthyroid and hypothyroid rats

 

Figure 7
View larger version (85K):
[in this window]
[in a new window]

  		Fig. 7. Subcellular distribution of the B1 subunit of the vacuolar H+-ATPase in the cortical collecting duct (CCD) at the light microscopic level. Kidneys were stained with antibodies against the B1 subunit of the vacuolar H+-ATPase (red) and calbindin D28k/aquaporin-2 (AQP2; combined together and shown in green). The arrow indicates a cell with apical staining, and the asterisk shows a cell with basolateral staining. A: CCD from an euthyroid rat. B: CCD from a hypothyroid rat. C: CCD from an euthyroid rat treated with NH4Cl. D: CCD from a hypothyroid rat treated with NH4Cl. Original magnification x400.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The ability of the kidney to maintain and regulate systemic acid-base homeostasis is under the control of a variety of systemic and local factors including several hormones such as angiotensin II, aldosterone, endothelin, and thyroid hormones (14, 19). The role of thyroid hormones in the stimulation of bicarbonate reabsorption in the proximal tubule and the expression of the brush-border membrane Na+/H+ exchanger NHE3 in growing and adult rats has been extensively described and analyzed (11, 18, 27, 28, 35). In addition, the role of thyroid hormones in the expression of the proximal tubular Na+-phosphate cotransporter NaPi-IIa has been described. A reduced expression of NaPi-IIa during hypothyroidism leads to renal phosphate wasting (2), where the increased phosphate load to the distal nephron could contribute to a higher proton buffering capacity and even stimulate renal acid clearance. On the other hand, hypothyroidism in humans has been reported to be associated in several cases with the development of renal tubular acidosis (13, 31).

We used a pharmacological model of mild hypothyroidism that has been characterized with respect to alterations in the renal expression of sodium- and water-transporting proteins (11, 35). Measurements of TSH and free T4 levels confirm the development of mild hypothyroidism in the group treated with methimazole, whereas substitution with L-thyroxine suppressed TSH upregulation and restored free T4 levels to the normal range as reported previously in rats (35, 41). The advantage of this pharmacological model compared with total thyroidectomy is evident from the fact that removal of the parathyroid glands together with the thyroid gland would also affect parathyroid hormone (PTH) levels. Chronic changes in PTH levels are associated with alterations in NHE3 and NaPi-IIa expression (17, 30), and direct effects of PTH on acid-base transport in the collecting duct have been reported (5, 21).

Mild hypothyroidism led to a strong reduction in NHE3 and NaPi-IIa expression as reported previously for models of total thyroidectomy (2, 4, 18, 35), confirming that the reduction in thyroid hormones was sufficient to alter biological processes. In addition to the previously described regulation of NHE3 and NaPi-IIa by thyroid hormones, we also found reduced expression of the electrogenic Na+-bicarbonate cotransporter KNBCe1 and the B2 subunit of the vacuolar H+-ATPase in the proximal tubule of HYPO rats. However, under control conditions HYPO rats did not display any signs of metabolic acidosis or urinary bicarbonate wasting despite the massive downregulation of NHE3 and kNBCe1. This is best explained by an upregulation of compensatory mechanisms in more distal parts of the nephron. Accordingly, increased expression of the chloride/bicarbonate exchanger AE1 expressed in type A intercalated cells was observed. Upregulation of AE1 has been reported in rat models of metabolic acidosis (20, 33), and loss of AE1 function is associated with distal renal tubular acidosis in humans (9, 22). The higher abundance of AE1 was not due to a change in the number of AE1-expressing cells as shown by analysis of relative cell numbers in the different segments of the collecting system. Interestingly, staining at the light microscopic level for the intercalated cell-specific B1 subunit of the vacuolar proton pump appeared as a rather cytosolic pattern in the cortical collecting duct of HYPO rats, which would indicate a possibly lower capacity to excrete protons. In the outer medullary collecting duct, no difference could be observed. It remains to be tested whether the apparent differences in staining are consistent with a change in the activity of proton pumps.

After induction of metabolic acidosis with oral NH4Cl loading, a more pronounced metabolic acidosis was observed in HYPO rats. An appropriate respiratory compensation was present in both EU and HYPO rats, as evident from low PCO2 values, ruling out an effect of hypothyroidism on central chemosensing and respiratory adaptation. The more severe metabolic acidosis was most likely not due to a renal failure to excrete acid, as urinary pH, urinary ammonia excretion, and net acid excretion were similar, if not even increased, in the HYPO group. Moreover, HYPO rats responded to acid loading with an upregulation of the kNBCe1 cotransporter as well as increased expression of the AE1 exchanger. Morphological analysis demonstrated that the higher AE1 abundance was associated with a higher relative number of AE1-expressing type A intercalated cells in the connecting tubule and cortical collecting duct. A similar remodeling of the collecting duct has been observed in various rat and mouse models of metabolic acidosis (1, 25, 40) and is thought to enhance the ability of the collecting duct to excrete acid and regenerate bicarbonate. Thus in HYPO rats lower expression of proteins involved in the initial step of bicarbonate reabsorption and proton secretion in the proximal tubule are compensated by upregulation of other acid-base transporters and remodeling of downstream nephron segments. The cause of the more severe metabolic acidosis in HYPO rats may lie in other systems that contribute to control of systemic acid-base balance. Along this line, during acidosis HYPO rats had mild diarrhea that may have led to intestinal bicarbonate losses, causing a subtraction acidosis.

In summary, mild hypothyroidism in rat causes downregulation of acid-base transporters in the proximal tubule and is associated with more severe metabolic acidosis after oral acid loading. However, metabolic acidosis is most likely of extrarenal origin. The kidney shows upregulation of compensatory mechanisms located in the collecting system such as activation of type A intercalated cells and remodeling of the collecting duct with increased numbers of acid-secretory intercalated cells.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by a fellowship of the Erwin-Riesch Foundation (to N. Mohebbi) and grants from the Swiss National Research foundation (31-109677/1) and the 6th Framework European Union Project EuReGene (to C. A. Wagner). The study was further supported by the University Research Priority Program of the University of Zurich.


Figure 8


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. A. Wagner, Institute of Physiology, Univ. of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland (e-mail: Wagnerca{at}access.unizh.ch)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Al-Awqati Q. Plasticity in epithelial polarity of renal intercalated cells: targeting of the H+-ATPase and band 3. Am J Physiol Cell Physiol 270: C1571–C1580, 1996.[Abstract/Free Full Text]
  2. Alcalde AI, Sarasa M, Raldua D, Aramayona J, Morales R, Biber J, Murer H, Levi M, Sorribas V. Role of thyroid hormone in regulation of renal phosphate transport in young and aged rats. Endocrinology 140: 1544–1551, 1999.[Abstract/Free Full Text]
  3. Bastani B, Purcell H, Hemken P, Trigg D, Gluck S. Expression and distribution of renal vacuolar proton-translocating adenosine triphosphatase in response to chronic acid and alkali loads in the rat. J Clin Invest 88: 126–136, 1991.[Web of Science][Medline]
  4. Baum M, Dwarakanath V, Alpern RJ, Moe OW. Effects of thyroid hormone on the neonatal renal cortical Na+/H+ antiporter. Kidney Int 53: 1254–1258, 1998.[CrossRef][Web of Science][Medline]
  5. Bengele HH, McNamara ER, Alexander EA. Effect of acute thyroparathyroidectomy on nephron acidification. Am J Physiol Renal Fluid Electrolyte Physiol 246: F569–F574, 1984.[Abstract/Free Full Text]
  6. Berthelot M. Violet d'aniline. Rep Chim App 1: 284, 1859.
  7. Biber J, Stieger B, Haase W, Murer H. A high yield preparation for rat kidney brush border membranes. Different behaviour of lysosomal markers. Biochim Biophys Acta 647: 169–176, 1981.[Medline]
  8. Bonnici B, Wagner CA. Postnatal expression of transport proteins involved in acid-base transport in mouse kidney. Pflügers Arch 448: 16–28, 2004.[CrossRef][Web of Science][Medline]
  9. Bruce LJ, Cope DL, Jones GK, Schofield AE, Burley M, Povey S, Unwin RJ, Wrong O, Tanner MJ. Familial distal renal tubular acidosis is associated with mutations in the red cell anion exchanger (Band 3, AE1) gene. J Clin Invest 100: 1693–1707, 1997.[Web of Science][Medline]
  10. Brungger M, Hulter HN, Krapf R. Effect of chronic metabolic acidosis on thyroid hormone homeostasis in humans. Am J Physiol Renal Physiol 272: F648–F653, 1997.[Abstract/Free Full Text]
  11. Cadnapaphornchai MA, Kim YW, Gurevich AK, Summer SN, Falk S, Thurman JM, Schrier RW. Urinary concentrating defect in hypothyroid rats: role of sodium, potassium, 2-chloride co-transporter, and aquaporins. J Am Soc Nephrol 14: 566–574, 2003.[Abstract/Free Full Text]
  12. Chan JC. The rapid determination of urinary titratable acid and ammonium and evaluation of freezing as a method of preservation. Clin Biochem 5: 94–98, 1972.[CrossRef][Web of Science][Medline]
  13. Drukker A, Dolberg U, Landau H. Renal tubular acidosis in a patient with hypothyroidism due to autoimmune thyroiditis - improvement with hormone replacement therapy. Int J Pediatr Nephrol 3: 205–209, 1982.[Web of Science][Medline]
  14. DuBose TD Jr, Alpern RJ. Renal tubular acidosis. In: The Metabolic and Molecular Bases of Inherited Disease (8th ed.), edited by Scriver CR, Beaudet AL, Sly WS, and Valle, D. New York: McGraw-Hill, 2001, p. 4983–5021.
  15. Duntas L, Roussel JP, Astier H, Keck FS, Rosenthal J, Pfeiffer EF. Aspects of chronic oral treatment with thyrotropin-releasing hormone: the hypothalamic-pituitary-thyroid axis in rats. A study with a pharmacological dose of thyrotropin-releasing hormone. Pharmacology 43: 106–112, 1991.[Web of Science][Medline]
  16. Finberg KE, Wagner CA, Stehberger PA, Geibel JP, Lifton RP. Molecular cloning and characterization of Atp6v1b1, the murine vacuolar H+-ATPase B1-subunit. Gene 318: 25–34, 2003.[CrossRef][Web of Science][Medline]
  17. Girardi AC, Titan SM, Malnic G, Reboucas NA. Chronic effect of parathyroid hormone on NHE3 expression in rat renal proximal tubules. Kidney Int 58: 1623–1631, 2000.[CrossRef][Web of Science][Medline]
  18. Gupta N, Dwarakanath V, Baum M. Maturation of the Na+/H+ antiporter (NHE3) in the proximal tubule of the hypothyroid adrenalectomized rat. Am J Physiol Renal Physiol 287: F521–F527, 2004.[Abstract/Free Full Text]
  19. Hamm LL, Alpern RJ. Cellular mechanisms of renal tubular acidification. In: The Kidney: Physiology and Pathophysiology (3rd ed.), edited by Seldin DW and Giebisch G. Philadelphia, PA: Lippincott Williams & Wilkins, 2000, p. 1935–1979.
  20. Huber S, Asan E, Jons T, Kerscher C, Puschel B, Drenckhahn D. Expression of rat kidney anion exchanger 1 in type A intercalated cells in metabolic acidosis and alkalosis. Am J Physiol Renal Physiol 277: F841–F849, 1999.[Abstract/Free Full Text]
  21. Hulter HN, Toto RD, Bonner EL, Ilnicki LP, Sebastian A. Renal and systemic acid-base effects of chronic hypoparathyroidism in dogs. Am J Physiol Renal Fluid Electrolyte Physiol 241: F495–F501, 1981.[Abstract/Free Full Text]
  22. Karet FE, Gainza FJ, Gyory AZ, Unwin RJ, Wrong O, Tanner MJ, Nayir A, Alpay H, Santos F, Hulton SA, Bakkaloglu A, Ozen S, Cunningham MJ, di Pietro A, Walker WG, Lifton RP. Mutations in the chloride-bicarbonate exchanger gene AE1 cause autosomal dominant but not autosomal recessive distal renal tubular acidosis. Proc Natl Acad Sci USA 95: 6337–6342, 1998.[Abstract/Free Full Text]
  23. Kumar V, Prasad R. Molecular basis of renal handling of calcium in response to thyroid hormone status of rat. Biochim Biophys Acta 1586: 331–343, 2002.[Medline]
  24. Loffing J, Loffing-Cueni D, Valderrabano V, Klausli L, Hebert SC, Rossier BC, Hoenderop JG, Bindels RJ, Kaissling B. Distribution of transcellular calcium and sodium transport pathways along mouse distal nephron. Am J Physiol Renal Physiol 281: F1021–F1027, 2001.[Abstract/Free Full Text]
  25. Madsen KM, Verlander JW, Kim J, Tisher CC. Morphological adaption of the collecting duct to acid-base disturbances. Kidney Int Suppl 33: S57–S63, 1991.[Medline]
  26. Michael UF, Chavez R, Cookson SL, Vaamonde CA. Impaired urinary acidification in the hypothyroid rat. Pflügers Arch 361: 215–220, 1976.[CrossRef][Web of Science][Medline]
  27. Michael UF, Kelley J, Meeks LA, Vaamonde CA. Renal acidification in the hypothyroid rat. Evaluation by urinary CO2 tension. Can J Physiol Pharmacol 59: 273–280, 1981.[Web of Science][Medline]
  28. Michael UF, Kelley J, Vaamonde CA. Impaired renal bicarbonate reabsorption in the hypothyroid rat. Am J Physiol Renal Fluid Electrolyte Physiol 236: F536–F540, 1979.[Abstract/Free Full Text]
  29. Morales MM, Brucoli HCP, Malnic G, Lopes AG. Role of thyroid hormones in renal tubule acidification. Mol Cell Biochem 154: 17–21, 1996.[Web of Science][Medline]
  30. Murer H, Hernando N, Forster I, Biber J. Proximal tubular phosphate reabsorption: molecular mechanisms. Physiol Rev 80: 1373–1409, 2000.[Abstract/Free Full Text]
  31. Oster JR, Michael UF, Perez GO, Sonneborn RE, Vaamonde CA. Renal acidification in hypothyroid man. Clin Nephrol 6: 398–403, 1976.[Medline]
  32. Prieto I, Segarra AB, Vargas F, Alba F, de Gasparo M, Ramirez M. Angiotensinase activity in hypothalamus and pituitary gland of hypothyroid, euthyroid and hyperthyroid adult male rats. Horm Metab Res 35: 279–281, 2003.[CrossRef][Web of Science][Medline]
  33. Sabolic I, Brown D, Gluck SL, Alper SL. Regulation of AE1 anion exchanger and H+-ATPase in rat cortex by acute metabolic acidosis and alkalosis. Kidney Int 51: 125–137, 1997.[Web of Science][Medline]
  34. Sagawa K, Murer H, Morris ME. Effect of experimentally induced hypothyroidism on sulfate renal transport in rats. Am J Physiol Renal Physiol 276: F164–F171, 1999.[Abstract/Free Full Text]
  35. Schmitt R, Klussmann E, Kahl T, Ellison DH, Bachmann S. Renal expression of sodium transporters and aquaporin-2 in hypothyroid rats. Am J Physiol Renal Physiol 284: F1097–F1104, 2003.[Abstract/Free Full Text]
  36. Seaton B, Ali A. Simplified manual high performance clinical chemistry methods for developing countries. Med Lab Sci 41: 327–336, 1984.[Web of Science][Medline]
  37. Slot C. Plasma creatinine determination. A new and specific Jaffé reaction method. Scand J Clin Lab Invest 17: 381–387, 1965.[Web of Science][Medline]
  38. Stehberger P, Schulz N, Finberg KE, Karet FE, Giebisch G, Lifton RP, Geibel JP, Wagner CA. Localization and regulation of the ATP6V0A4 (a4) vacuolar H+-ATPase subunit defective in an inherited form of distal renal tubular acidosis. J Am Soc Nephrol 14: 3027–3038, 2003.[Abstract/Free Full Text]
  39. Stehberger PA, Shmukler BE, Stuart-Tilley AK, Peters LL, Alper SL, Wagner CA. Distal renal tubular acidosis in mice lacking the AE1 (band3) Cl–/HCO3– exchanger (slc4a1). J Am Soc Nephrol 18: 1408–1418, 2007.[Abstract/Free Full Text]
  40. Wagner CA, Finberg KE, Stehberger PA, Lifton RP, Giebisch GH, Aronson PS, Geibel JP. Regulation of the expression of the Cl–/anion exchanger pendrin in mouse kidney by acid-base status. Kidney Int 62: 2109–2117, 2002.[CrossRef][Web of Science][Medline]
  41. Zandieh-Doulabi B, Platvoet-ter Schiphorst M, Kalsbeek A, Wiersinga WM, Bakker O. Hyper and hypothyroidism change the expression and diurnal variation of thyroid hormone receptor isoforms in rat liver without major changes in their zonal distribution. Mol Cell Endocrinol 219: 69–75, 2004.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
J.-L. Horng, L.-Y. Lin, and P.-P. Hwang
Functional regulation of H+-ATPase-rich cells in zebrafish embryos acclimated to an acidic environment
Am J Physiol Cell Physiol, April 1, 2009; 296(4): C682 - C692.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
J. Gattineni, D. Sas, A. Dagan, V. Dwarakanath, and M. Baum
Effect of thyroid hormone on the postnatal renal expression of NHE8
Am J Physiol Renal Physiol, January 1, 2008; 294(1): F198 - F204.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/1/F416    most recent
00391.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mohebbi, N.
Right arrow Articles by Wagner, C. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mohebbi, N.
Right arrow Articles by Wagner, C. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.