Deletion of the Cl-/HCO3- exchanger pendrin downregulates calcium-absorbing proteins in the kidney and causes calcium wasting.
ABSTRACT The epithelial calcium channel (ECaC) (TRPV5) and the Cl-/HCO3- exchanger pendrin (SLC26A4) are expressed on the apical membrane of tubular cells in the distal nephron and play essential roles in calcium re-absorption and bicarbonate secretion, respectively, in the kidney.
A combination of functional and molecular biology techniques were employed to examine the role of pendrin deletion in calcium excretion.
Here, we demonstrate that deletion of pendrin causes acidic urine [urine pH 4.9 in knockout (KO) versus 5.9 in wild-type (WT) mice, P<0.03)] and downregulates the calcium-absorbing molecules ECaC and Na/Ca exchanger in the kidney, as shown by northern hybridization, immunoblot analysis and/or immunofluorescent labeling. These changes were associated with a ∼100% increase in 24-h urine calcium excretion in pendrin null mice. Subjecting the pendrin WT and KO mice to oral bicarbonate loading for 12 days increased the urine pH to ∼8 in both genotypes, normalized the expression of ECaC and Na/Ca exchanger and reduced the urine calcium excretion in pendrin-null mice to levels comparable to WT mice.
We suggest that pendrin dysfunction should be suspected and investigated in humans with an otherwise unexplained acidic urine and hypercalciuria.
- SourceAvailable from: Kamran Ahmed[show abstract] [hide abstract]
ABSTRACT: Cystinuria is an autosomal recessive disorder in renal tubular and intestinal transport of dibasic amino acids, which results in increased urinary excretion of cystine, ornithine, lysine and arginine. It affects 1 in 20 000 people and is caused by a defect in the rBAT gene on chromosome 2. Development of urinary tract cystine calculi is the only clinical manifestation of this disease. Owing to recurrent episodes of stone formation, these patients require a multi-modal approach to management. The role of medical management and minimally invasive surgery was reviewed for the treatment of cystinuria.Postgraduate medical journal 01/2007; 82(974):799-801. · 1.38 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Pendrin belongs to a superfamily of Cl-/anion exchangers and is expressed in the inner ear, the thyroid gland, and the kidney. In humans, mutations in pendrin cause Pendred syndrome characterized by sensorineural deafness and goiter. Recently pendrin has been localized to the apical side of non-type A intercalated cells of the cortical collecting duct, and reduced bicarbonate secretion was demonstrated in a pendrin knockout mouse model. To investigate a possible role of pendrin in modulating acid-base transport in the cortical collecting duct, we examined the regulation of expression of pendrin by acid-base status in mouse kidney. Mice were treated orally either with an acid or bicarbonate load (0.28 mol/L NH4Cl or NaHCO3) or received a K+-deficient diet for one week. Immunohistochemistry and Western blotting was performed. Acid-loading caused a reduction in pendrin protein expression levels within one day and decreased expression to 23% of control levels after one week. Concomitantly, pendrin protein was shifted from the apical membrane to the cytosol, and the relative abundance of pendrin positive cells declined. Similarly, in chronic K+-depletion, known to elicit a metabolic alkalosis, pendrin protein levels decreased and pendrin expression was shifted to an intracellular pool with the relative number of pendrin positive cells reduced. In contrast, following oral bicarbonate loading pendrin was found exclusively in the apical membrane and the relative number of pendrin positive cells increased. These results are in agreement with a potential role of pendrin in bicarbonate secretion and regulation of acid-base transport in the cortical collecting duct.Kidney International 01/2003; 62(6):2109-17. · 7.92 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The basolateral Cl(-)/HCO(3)(-) exchanger in parietal cells plays an essential role in gastric acid secretion mediated via the apical gastric H(+)-K(+)-ATPase. Here, we report the identification of a new Cl(-)/HCO(3)(-) exchanger, which shows exclusive expression in mouse stomach and kidney, with expression in the stomach limited to the basolateral membrane of gastric parietal cells. Tissue distribution studies by RT-PCR and Northern hybridizations demonstrated the exclusive expression of this transporter, also known as SLC26A7, to stomach and kidney, with the stomach expression significantly more abundant. No expression was detected in the intestine. Cellular distribution studies by RT-PCR and Northern hybridizations demonstrated predominant localization of SLC26A7 in gastric parietal cells. Immunofluorescence labeling localized this exchanger exclusively to the basolateral membrane of gastric parietal cells, and functional studies in oocytes indicated that SLC26A7 is a DIDS-sensitive Cl(-)/HCO(3)(-) exchanger that is active in both acidic and alkaline pH(i). On the basis of its unique expression pattern and function, we propose that SLC26A7 is a basolateral Cl(-)/HCO(3)(-) exchanger in gastric parietal cells and plays a major role in gastric acid secretion.AJP Gastrointestinal and Liver Physiology 07/2003; 284(6):G1093-103. · 3.65 Impact Factor
Nephrol Dial Transplant (2012) 27: 1368–1379
Advance Access publication 26 August 2011
Deletion of the Cl2/HCO32exchanger pendrin downregulates
calcium-absorbing proteins in the kidney and causes calcium wasting
Sharon Barone1,2,3, Hassane Amlal1,2, Jie Xu1,2,3and Manoocher Soleimani1,2,3
1Research Services, Veterans Administration Medical Center, Cincinnati, OH, USA,2Department of Medicine, University of Cincinnati,
Cincinnati, OH, USA and3Center on Genetics of Transport and Epithelial Biology, University of Cincinnati, Cincinnati, OH, USA
Correspondence and offprint requests to: Manoocher Soleimani; E-mail: firstname.lastname@example.org
Background. The epithelial calcium channel (ECaC)
are expressed on the apical membrane of tubular cells in
the distal nephron and play essential roles in calcium
re-absorption and bicarbonate secretion, respectively, in the
Methods. A combination of functional and molecular biol-
deletion in calcium excretion.
Results. Here, we demonstrate that deletion of pendrin
causes acidic urine [urine pH 4.9 in knockout (KO) versus
5.9 in wild-type (WT) mice, P < 0.03)] and downregulates
the calcium-absorbing molecules ECaC and Na/Ca ex-
changer in the kidney, as shown by northern hybridization,
immunoblot analysis and/or immunofluorescent labeling.
These changes were associated with a ~100% increase in
24-h urine calcium excretion in pendrin null mice. Subject-
for 12 days increased the urine pH to ~8 in both genotypes,
normalized the expression of ECaC and Na/Ca exchanger
and reduced the urine calcium excretion in pendrin-null
mice to levels comparable to WT mice.
Conclusions. We suggest that pendrin dysfunction should
be suspected and investigated in humans with an otherwise
unexplained acidic urine and hypercalciuria.
Keywords: acid-base transporters; acidic urine; calcium excretion; distal
nephron; urine alkalinization
SLC26 proteins are members of a conserved family of anion
transporters, which display distinct and/or limited tissue ex-
ing chloride, bicarbonate, sulfate and oxalate, with variable
specificity [9, 10] and show a specific cellular or subcellular
localization pattern [1–8]. Several SLC26A members can
function predominantly as chloride/bicarbonate exchangers,
including SLC26A3 (DRA) and SLC26A4 (pendrin) [9–16].
Genes coding for these transporters are mapped to chromo-
some7andare arrangedina headtoheadorientation[11–13].
Other members such as SLC26A6 (PAT1), SLC26A7 and
SLC26A9 can function in multiple anion exchange modes,
including chloride/bicarbonate exchange [14–20]. SLC26A7
and SLC26A9 can also function as chloride channels [20–23].
Pendrin or SLC26A4 is abundantly expressed in the thy-
roid, inner ear and the kidney [4, 12, 24–26]. Pendrin ex-
pression in the kidney is limited to the apical membrane of
non-A-intercalated cells in the cortical collecting duct
(CCD), connecting tubules and the distal convoluted tu-
bules [12, 24–26] and plays an important role in bicarbon-
ate secretion in the distal nephron [24, 27]. Animals lacking
pendrin produce very acidic urine as a result of decreased
apical Cl?/HO3?exchanger activity in their CCDs [27, 28].
The bulk of filtered calcium is reabsorbed in the proximal
tubule and the thick ascending limb of Henle’s loop via a
passive paracellular pathway [29, 30]. Calcium delivered to
the distal nephron is reabsorbed through an active transcel-
lular pathway that includes epithelial calcium channel
(ECaC), calbindin and basolateral Na/Ca exchanger acting
inseries[31–39]. ECaC, alsoknown asTRPV5,is expressed
on the apical membrane of epithelial cells in the distal con-
voluted and connecting tubules of the kidney [31–33]. Down-
regulation or ablation of ECaC has been associated with
profound calcium wasting by the kidney, indicating that this
molecule is essential for calcium re-absorption in the distal
nephron [34, 35]. ECaC is known to be inhibited by testoster-
one and extracellular acidic pH [35–37]. In addition to the
apical ECaC, the cytoplasmic calcium-binding protein calbin-
din carries calcium ions from the apical to the basolateral side
of the cells, while the basolateral Na/Ca exchanger mediates
pathway plays an important role in vectorial re-absorption of
calcium in the distal nephron [38, 39].
Given the important role of pendrin in urinary pH regu-
lation, we sought to examine the impact of pendrin ablation
on the rate of urinary calcium excretion and the expression
of the calcium-absorbing transport proteins in the distal
? The Author 2011. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
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nephron. Our studies demonstrate that the expression of
calcium-absorbing pathway molecules (apical ECaC and
basolateral Na/Ca exchanger) is downregulated in pendrin
knockout (KO) mice. These changes were associated with a
significant renal calcium wasting. We further demonstrate
that urine alkalinization in pendrin KO mice increased the
expression of calcium-absorbing molecules and reduced
calcium excretion to levels observed in wild-type (WT)
mice. The significance of the results will be discussed.
Materials and methods
Mice were cared for in accordance with protocols approved by the Institu-
tional Animal Care and Use Committee (IACUC) at the University of
Cincinnati. All animal handlers are IACUC trained. Pendrin KO (Pds?/?)
and WT (Pds1/1) mice were used for these studies. Animals were allowed
free access to water and food. The use of anesthetics (pentobarbital sodium)
and the method of euthanasia (pentobarbital sodium overdose) were
approved according to the institutional guidelines.
Urine alkalinization was performed by subjecting Pds1/1and Pds?/?
mice to oral sodium bicarbonate (280 mM) added to their drinking water
for 12 days. In separate studies, animals were placed in metabolic cages,
subjected to 100 mM oral bicarbonate and received daily acetazolamide
(ACTZ), a carbonic anhydrase inhibitor, at 20 mg/kg/day subcutaneously
for 4 days, to ensure the generation of alkaline urine pH and prevent the
induction of metabolic acidosis by ACTZ, which could downregulate
calcium absorbing molecules in the distal nephron.
Genotyping of Pds1/1and Pds?/?mice
The genotype of the pups was determined by polymerase chain reaction
(PCR) amplification and electrophoretic analysis of DNA extracted from
their tail clippings as previously described . The PCR reaction on
isolated tail DNA to identify WT mice was performed using the following
primers: 5#-AGGTAAGATGCTGCTGGATAGG-3# (forward) and 5#-
GCAGGCAAGCATTCTACCAC-3# (reverse), which amplify a 1.9-kb
band. The PCR reaction to identify KO mice was performed using the
(forward) and 5#-GGCAGGCAAGCATTCTACCACTAAG-3# (reverse),
which amplify a 1.8-kb band. The PCR conditions were as follows: Seg-
ment 1, 2 min at 94?C (denature) 1 cycle; Segment 2, 35 cycles of 30 s at
94?C (denature), 30 s at 65?C (annealing), 2 min at 68?C (extension) and
Segment 3, link to 68?C for 5 min (1 cycle).
RNA isolation and northern blot hybridization
Total cellular RNA was extracted from mouse kidney cortex and medulla
according to established methods, quantitated spectrophotometrically and
stored at ?80?C. Total RNA samples (30 lg per lane) were fractionated on
a 1.2% agarose–formaldehyde gel, transferred to Magna NT nylon mem-
branes, cross-linked by ultraviolet light and baked.32P-labeled rat (or
mouse) probes were used for northern blot analyses. Complementary
DNA (cDNA) fragments spanning nucleotides1148–1586 of ECaC (ac-
cession number AF209196), nucleotides 120–629 of calbindin (accession
number NM031984) and nucleotides 1949–2812 of Na/Ca exchanger (ac-
cession number NM019268) were used as gene-specific probes. A mouse
cDNA fragment spanning nucleotides 1883–2217 of pendrin was used for
northern hybridization. Hybridization was performed according to estab-
lished methods. The membranes were washed, blotted dry and exposed to
a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA). The
signal strength of hybridization bands was quantitated by densitometry
using ImageQuaNT software (Molecular Dynamics).
Membrane proteins isolated from the mouse kidney cortex were size frac-
tionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(30 lg per lane) and transferred to nitrocellulose membrane. The membrane
was blocked with 5% milk proteins and then incubated for 6 h with desired
concentrations of specific antibodies. The secondary antibody was a donkey
anti-rabbit IgG conjugated to horseradish peroxidase (Pierce, Rockford, IL)
for polyclonal antibodies and a goat anti-mouse IgG conjugated to horse-
radish peroxidase for monoclonal antibodies. The recognized bands (pro-
teins) were visualized using the chemiluminescence method (RapidStep
ECL Reagent, San Diego, CA) and captured on light-sensitive imaging film
(MidSci, St Louis, MO). The antibodies utilized for western blot analyses
were pendrin , Na/Ca exchanger (Abcam, Cambridge, MA), ECaC
(Novus Biologicals, Littleton, CO) and calbindin (Abcam). The dilutions
for ECaC, calbindin and basolateral Na/Ca exchanger antibodies were 1/
600, 1/800 and 1/1000, respectively.
Immunofluorescence labeling studies
Mice were euthanized with an overdose of pentobarbital sodium and per-
fused through the left ventricle with 0.9% saline followed by cold 4%
paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). Kidneys
were removed, cut in tissue blocks, fixed in formaldehyde solution over-
night at 4?C and then transferred to 30% sucrose in 0.1 M sodium phos-
phate buffer (pH 7.4) at 4?C. The tissues were frozen in liquid nitrogen,
and 6-lm sections were cut usinga cryostat. Frozensections were stored at
?80?C until used. Single immunofluorescence labeling was performed as
described [16–18] using either Alexa Fluor 488 (green) or Alexa Fluor 594
(red) antibody (Invitrogen, Carlsbad, CA) as secondary antibodies. For
single-labeling studies with pendrin antibody, we utilized Alexa Fluor
594-conjugated (red color) goat anti-mouse IgG labeling kit.
For immunofluorescence double labeling, polyclonal antibodies for
pendrin (at 1:25 dilution) or calbindin (at 1:50 dilution) were used in
conjunction with the monoclonal Na/Ca exchanger antibodies (1:100 di-
lution). Pendrin or calbindin antibodies were detected by Alexa Fluor 488-
conjugated (green color) anti-rabbit IgG labeling kit and Na/Ca exchanger
antibodies were detected using Alexa Fluor 594-conjugated (red color)
goat anti-mouse IgG labeling kit (Invitorgen Molecular probes, Eugene,
OR) according to the manufacturer’s instructions.
For immunofluorescent microscopy studies, paraffin-embedded slides
were subjected to antigen retrieval protocol. Frozen kidney sections were
allowed to thaw at room temperature and were subsequently rehydrated in
phosphate-buffered saline (PBS) for 15 min and permeabilized in PBS con-
taining 0.3% Triton X-100 [Phosphate Buffered saline with Triton (PBT)]
for 20 min at room temperature. Non-specific binding was blocked with
Image-iT FX signal enhancer (Invitrogen) for 30 min at room temperature.
Primaryantibodiesinadiluentof 0.3%TritonX-100and10% bovineserum
albumin in 0.1 M PBS were applied to the sections overnight at 4?C in a
humidified chamber. Sections underwent three PBS washes of 10 min each
on the orbital shaker. Sections were then briefly allowed to dry and cover-
slipped with the anti-fade fluorescent mounting medium (Vectashield, Bur-
lingame, CA). Sections were examined andimageswere acquired on a Zeiss
Axio-plan fluorescent microscope.
Balanced studies in experimental animals
Mice were housed in metabolic cages and had free access to rodent chow
and water. Food intake, water intake and urine volume were measured daily.
Urine was collected under mineral oil. Urine calcium concentration was
measured via a Calcium Assay Kit (BioChain Institute, Hayward, CA).
Serum Ca21concentration was measured with an i-STAT?-1 analyzer us-
ing i-STAT EG71 cartridges (Abbott Laboratories, Abbott Park, IL).
[32P]dCTP was purchased from Perkin Elmer (Shelton, CT). Nitrocellu-
lose filters and other chemicals were purchased from Sigma (St Louis,
MO). Probes were labeled with [32P]dCTP via QIAquick Nucleotide Re-
moval Kit (Qiagen, Valencia, CA).
The results for northernhybridization,western blotting, calcium excretion or
urine pH studies are expressed as means ? SE. Statistical significance
between various experimental groups was determined by Student’s unpaired
t-test or analysis of variance, and P <0.05 was considered significant.
The generation of Pds?/?mice used in these studies was
recently reported from our laboratory . Figure 1a is a
representative genotype analysis by PCR on tail DNA and
depicts the identification of WT, heterozygote and Pds?/?
Pendrin, calcium excretion and kidney stone1369
mice. The sequences of primers used in genotyping are
included in Materials and methods.
Northern blot analysis and immunofluorescent detection
of pendrin in the kidneys of Pds1/1and Pds?/?mice
Northern blot analysis results indicate the complete absence of
mice (Figure 1b). Immunofluorescent labeling with pendrin
antibodies demonstrated its specific localization to the apical
membrane of cells in CCD in Pds1/1mice. Pendrin staining
was not detected in CCD cells of Pds?/?mice (Figure 1c).
Localization of pendrin and calcium-absorbing
molecules in the distal nephron
Published reports indicate that Pds?/?mice have signifi-
cantly lower urine pH compared to their Pds1/1littermates
Fig. 1. Genotyping and expression of pendrin in Pds1/1and Pds?/?mice. (a) Genotyping of Pds1/1and Pds?/?mice. A representative ethidium
bromide staining of agarose gel demonstrates the identification of wild-type (Pds1/1), heterozygote (Pds1/?) and pendrin-deficient (Pds?/?) mice. (b)
Northern blot analysis. Pendrin mRNA was abundantly expressed in the kidney cortices of Pds1/1mice but was absent in Pds?/?mice. The faint larger
band in Pds?/?mice is the neocassette-containing untranslatable mutant transcript (see immunolabeling studies below). (c). Immunofluorescent labeling
of pendrin in kidneys of Pds1/1and Pds?/?mice. Immunofluorescence labeling with pendrin-specific antibodies (red color) detected the expression of
pendrin on the apical membrane of cells in the CCD in WT mice but did not detect any expression in pendrin KO mice.
1370S. Barone et al.
[27, 28]. In the next series of experiments, we examined
possible co-localization of pendrin and calcium-absorbing
molecules in the distal nephron. Toward this end, double
immunofluorescent labeling studies were performed using
pendrin and Na/Ca exchanger antibodies. As demonstrated
in Figure 2a and b (merged images in middle panels), the
Na/Ca exchanger and pendrin show distinct localization
patterns. In distal convoluted tubules and connecting tu-
bules (Figure 2a and b, see yellow and white arrows), the
two transporters are expressed by distinct cell types within
the same segment (Figure 2a and b). These results suggest
that distal convoluted and connecting tubules express both
pendrin and calcium absorbing molecules, with pendrin
appearing on the apical membrane of B (should this be
b)-intercalated cells and Na/Ca exchanger localizing to
the basolateral membrane of non-intercalated cells. In ad-
dition, pendrin labeling is also detected on the apical mem-
brane of B-intercalated cells of CCD, which do not express
any Na/Ca exchanger (Figure 2b).
Given the localization of pendrin and calcium absorbing
molecules to the distal convoluted and connecting tubules
(above) and given the role of luminal pH in regulating ECaC
expression and/or activity [36, 37], we entertained the pos-
sibility that the acidic urine pH in pendrin KO (Pds?/?) mice
might downregulate ECaC activity in the distal nephron,
therefore decreasing calcium re-absorption. To test this pos-
sibility, animals were placed in metabolic cages and their
water and food intake as well as body weight and urine
output was recorded on a daily basis (Figure 3a). After ac-
climatization, daily urine output was collected and analyzed.
Urine pH was determined with a microelectrode and urine
calcium was measured with a calcium assay kit (Materials
and methods). As shown in Figure 3b (top panel), urine
calcium excretion was significantly increased in pendrin
KO mice (P < 0.05 versus WT mice). The increase in urine
calcium excretion was paralleled by a reduction in urine pH
in pendrin KO mice (Figure 3b, bottom panel).
Effect of pendrin ablation on the expression of ECaC,
Na/Ca exchanger and calbindin
Given the increased urine calcium excretion in pendrin KO
mice (Figure 3), we examined the expression of ECaC, the
apical calcium-absorbing channel in the distal nephron
[29–32]. The ECaC mRNA expression and protein abun-
dance decreased significantly in the kidney cortex of Pds?/?
mice (Figure 4a and b). Northern blot analysis of RNA
isolated from the kidneys of four separate animals showed
that the expression of ECaC transcript decreased by 58% in
the cortices of Pds?/?mice (right panel in Figure 4a) (P <
0.05 versus Pds1/1). The protein levels of ECaC were also
diminished by ~45% in Pds?/?mice (right panel in Figure
4b) (P < 0.05 versus Pds1/1). Our attempts at obtaining
immunofluorescent labeling of ECaC in the kidneys of exper-
imental animals were not successful.
The apical ECaC works in tandem with the cytoplasmic
calbindin and the basolateral Na/Ca exchanger to reabsorb
calcium in the distal nephron. In the next series of experi-
ments, we examined the mRNA expression and protein
abundance of Na/Ca exchanger (Figure 5A) and calbindin
(Figure 5B) in the kidneys of Pds1/1and Pds?/?mice. Our
results demonstrated that the mRNA expression of the baso-
lateral Na/Ca exchanger was significantly reduced in the
kidneys of Pds?/?mice (Figure 5A, a) (P < 0.05 versus
Pds1/1). Immunofluorescent labeling on kidney sections
and western blot analysis on membrane proteins showed
significant reduction in the expression of the Na/Ca ex-
changer in Pds?/?mice (Figure 5A, b and c).
Northern blot analyses further indicated that the expres-
sion of calbindin mRNA was reduced in the kidneys of
Pds?/?mice versus Pds1/1mice (Figure 5B, a; P <
0.05). Immunofluorescent labeling studies on kidney sec-
tions (Figure 5B, b) and western blot analysis on membrane
proteins showed significant reduction in the expression of
calbindin in Pds?/?mice (Figure 5B, c).
Effect of urine alkalinization on calcium excretion and
the expression of calcium-reabsorbing molecules
The purpose of the next series of experiments was to exam-
ine the role of urine pH on the expression of calcium-absorb-
ing molecules in the kidney and its impact on urine calcium
excretion. Toward this end, Pds1/1and Pds?/?mice were
placed in metabolic cages and after acclimatization on
normal food and water for 3 days were switched to oral
sodium bicarbonate (280 mM) added to their drinking water.
Animals were maintained on oral bicarbonate for 12 days.
The water intake, urine output, food intake and body weight
were measured daily. Figure 6a shows body weight, food
intake, water intake and urine output at baseline and in
response to oral bicarbonate loading. As shown, animals
on bicarbonate solution maintained their food intake but
increased their water intake and urine output. Figure 6b de-
picts calcium excretion rate and urine pH in WT and Pds?/?
mice at baseline and in response to bicarbonate loading. As
indicated, urinepHwassignificantly lowerinPds?/?mice at
baseline state compared to Pds1/1mice (Figure 6b, bottom
panel). Urine pH increased in both Pds1/1and Pds?/?mice
immediately following switching to oral bicarbonate therapy
and reached comparable values in both genotypes on Days
11 and 12 (Figure 6b, bottom panel).
The 24-h calcium excretion rate in Pds?/?mice was in-
creased at basal state but decreased significantly in response
to oral bicarbonate loading. In Pds1/1mice, urine calcium
excretion was lower at basal state as compared to Pds?/?
mice, confirming the studies in Figure 3. The calcium ex-
cretionin Pds?/?miceonoral bicarbonate loadingdecreased
to values that were comparable to Pds1/1mice on the same
treatment (Figure 6b, top panel).
anhydrase inhibitor, has several distinct effects on urinary
parameters, acid-base status and calcium excretion . It
results in bicarbonate wasting, which is manifested by urine
alkalinization but also causes metabolic acidosis. In the next
series of experiments, Pds?/?mice were placed on 100 mM
oral sodium bicarbonate added to their drinking water and
received daily subcutaneous doses of ACTZ, a carbonic anhy-
concentration was 29.2 ? 0.6 mEq/L, which is not signifi-
Pendrin, calcium excretion and kidney stone1371
Fig. 2. Localization of pendrin and Na/Ca exchanger in the kidney. (a) Co-localization of pendrin and Na/Ca exchanger in the connecting tubule.
Immunofluorescent labeling studies showed the expression of pendrin (green) and Na/Ca exchanger (red) in the same tubules (connecting tubules) but in
distinct cells (white arrows for pendrin and yellow arrows for Na/Ca exchanger). (b). Localization of pendrin and Na/Ca exchanger in the distal nephron.
Immunofluorescent labeling studies showed the co-localization of pendrin (green color) and Na/Ca exchanger (red color) in the same distal tubules and
connecting tubules but in distinct cells (white arrows for pendrin and yellow arrows for Na/Ca exchanger). Pendrin is also expressed in the collecting duct
(arrows), whereas Na/Ca exchanger is not.
1372S. Barone et al.