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.
- [Show abstract] [Hide abstract]
ABSTRACT: The phylogenetically ancient SLC26 gene family encodes multifunctional anion exchangers and anion channels transporting a broad range of substrates, including Cl(-), HCO3(-), sulfate, oxalate, I(-), and formate. SLC26 polypeptides are characterized by N-terminal cytoplasmic domains, 10-14hydrophobic transmembrane spans, and C-terminal cytoplasmic STAS domains, and appear to be homo-oligomeric. SLC26-related SulP proteins of marine bacteria likely transport HCO3(-) as part of oceanic carbon fixation. SulP genes present in antibiotic operons may provide sulfate for antibiotic biosynthetic pathways. SLC26-related Sultr proteins transport sulfate in unicellular eukaryotes and in plants. Mutations in three human SLC26 genes are associated with congenital or early onset Mendelian diseases: chondrodysplasias for SLC26A2, chloride diarrhea for SLC26A3, and deafness with enlargement of the vestibular aqueduct for SLC26A4. Additional disease phenotypes evident only in mouse knockout models include oxalate urolithiasis for Slc26a6 and Slc26a1, non-syndromic deafness for Slc26a5, gastric hypochlorhydria for Slc26a7 and Slc26a9, distal renal tubular acidosis for Slc26a7, and male infertility for Slc26a8. STAS domains are required for cell surface expression of SLC26 proteins, and contribute to regulation of the cystic fibrosis transmembrane regulator in complex, cell- and tissue-specific ways. The protein interactomes of SLC26 polypeptides are under active investigation.Molecular Aspects of Medicine 04/2013; 34(2-3):494-515. · 10.30 Impact Factor
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ABSTRACT: Tenofovir disoproxil fumarate, the prodrug of nucleotide reverse transcriptase inhibitor tenofovir, shows high efficacy and relatively low toxicity in HIV patients. However, long-term kidney toxicity is now acknowledged as a modest but significant risk for tenofovir-containing regimens, and continuous use of tenofovir in HIV therapy is currently under question by practitioners and researchers. Co-morbidities (hepatitis C, diabetes), low body weight, older age, concomitant administration of potentially nephrotoxic drugs, low CD4 count, and duration of therapy are all risk factors associated with tenofovir-associated tubular dysfunction. Tenofovir is predominantly eliminated via the proximal tubules of the kidney, therefore drug transporters expressed in renal proximal tubule cells are believed to influence tenofovir plasma concentration and toxicity in the kidney. We review here the current evidence that the actions, pharmacogenetics, and drug interactions of drug transporters are relevant factors for tenofovir-associated tubular dysfunction. The use of creatinine and novel biomarkers for kidney damage, and the role that drug transporters play in biomarker disposition, are discussed. The lessons learnt from investigating the role of transporters in tenofovir kidney elimination and toxicity can be utilized for future drug development and clinical management programs.Frontiers in Pharmacology 11/2014; 5:248.
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ABSTRACT: Solute-linked carrier 26 (SLC26) isoforms constitute a conserved family of anion transporters with 10 distinct members. Except for SLC26A5 (prestin), all can operate as multifunctional anion exchangers, with three members (SLC26A7, SLC26A9, and SLC26A11) also capable of functioning as chloride channels. Several SLC26 isoforms can specifically mediate Cl(-)/HCO3(-) exchange. These include SLC26A3, A4, A6, A7, A9, and A11, which are expressed in the kidney except for SLC26A3 (DRA), which is predominantly expressed in the intestine. SLC26 Cl(-)/HCO3(-) exchanger isoforms display unique nephron segment distribution patterns with distinct subcellular localization in the kidney tubules. Together with studies in pathophysiologic states and the examination of genetically engineered mouse models, the evolving picture points to important roles for the SLC26 family in health and disease states. This review summarizes recent advances in the characterization of the SLC26 Cl(-)/HCO3(-) exchangers in the kidney with emphasis on their essential role in diverse physiological processes, including chloride homeostasis, oxalate excretion and kidney stone formation, vascular volume and blood pressure regulation, and acid-base balance.Kidney International advance online publication, 1 May 2013; doi:10.1038/ki.2013.138.Kidney International 05/2013; · 8.52 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: email@example.com
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.
1370 S. 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.
1372 S. Barone et al.
basal conditions . Urine pH was 8.2 ? 0.3 in Pds?/?mice
comparable to oral bicarbonate loading alone in the same
genotype (Figure 6b) but is significantly higher than basal
conditions in Pds?/?mice (Figures 3 and 6b and c). Calcium
excretion was mildly reduced in Pds?/?mice treated with
Fig. 3. Effect of pendrin ablation on urine pH and calcium excretion. (a). Balanced studies in Pds1/1and Pds?/?mice. Body weight, food intake and
urine output were measured at baseline state in animals in metabolic cages. (b). Urine pH and calcium excretion in Pds1/1and Pds?/?mice. Urine pH
was determined by microelectrode in Pds1/1and Pds?/?mice. As shown, urine pH (lower panel) was significantly decreased and calcium excretion (top
panel) was significantly increased in Pds?/?mice at baseline.
Pendrin, calcium excretion and kidney stone1373
bicarbonate and ACTZ versus basal conditions (Figure 6c,
right panel) but remained significantly higher relative to oral
bicarbonate loading alone (Figure 6b).
Finally, we examined the effect of urine alkalinization on
Toward that end, WT (Pds1/1) and pendrin KO (Pds?/?)
mice on oral bicarbonate solution for 12 days were euthan-
ized and their kidneys were examined for the expression of
ECaC, calbindin and Na/Ca exchanger. As shown in
Figure 7A, a, mRNA levels of kidney ECaC in Pds?/?mice
receiving oral bicarbonate were comparable to Pds1/1mice
subjected to the same treatment. Furthermore, western blot
analyses indicated that the ECaC levels in Pds?/?mice on
oral bicarbonate loading became comparable to those ob-
served in Pds1/1mice (Figure 7A, b).
The expression levels of basolateral Na/Ca exchanger and
calbindin were examined in animals subjected to bicarbonate
loading. Our results indicate that the expression of Na/Ca
exchanger mRNA and protein in the kidneys of Pds?/?mice
receiving oral bicarbonate increased to levels comparable to
expression and protein levels of calbindin in Pds?/?mice on
oral bicarbonate were also elevated to levels comparable to
those of the Pds1/1mice (Figure 7C, a and b).
The vectorial re-absorption of calcium in the distal nephron
is mediated via ECaC, calbindin and Na/Ca exchanger
working in sequence. The apical calcium re-absorption is
the rate-limiting step in this process and is mediated via
ECaC [31–35]. Downregulation or ablation of ECaC has
been associated with profound renal calcium wasting, in-
dicating the important role that this channel plays in cal-
cium re-absorption in the kidney [34, 35].
Pendrin or SLC26A4, which functions in Cl?/HO3?ex-
change mode in the kidney, is located on the apical mem-
brane of non-A (B and non-A, non-B)-intercalated cells in
the CCD, connecting tubule and the distal convoluted tu-
bule [24–26] and is the major transporter responsible for
bicarbonate secretion in the distal nephron [24, 27]. Pen-
drin shows adaptive downregulation during metabolic
acidosis and potassium depletion and upregulation in re-
sponse to bicarbonate loading and aldosterone treatment
The present studies establish a novel interaction between
the pendrin- and the calcium-absorbing pathway in the
distal nephron. Our studies (Figure 2) demonstrate that
the ablation of pendrin downregulates the expression of
ECaC and the protein that regulates the rate-limiting step
in calcium re-absorption pathway in the distal nephron. As
a result, Pds?/?mice develop calcium wasting (Figure 3).
Indeed, the 24-h calcium excretion rate increased by
~100% in Pds?/?mice (Figure 3). In addition to ECaC,
pendrin deletion downregulates the cytoplasmic calbindin
and the basolateral Na/Ca exchanger in the distal nephron
(Figure 5). Our results demonstrate that the decrease in the
mRNA levels of ECaC, calbindin and Na/Ca exchanger
correlates with a reduction in their protein abundance and
increased urinary calcium wasting in Pds?/?mice.
ECaC expression and/or activity are shown to be regu-
lated by extracellular pH in in vitro systems [36, 37]. In-
deed, it has been shown that ECaC expression is reduced in
an acidic extracellular environment; whereas, its cell sur-
face expression increases in an alkaline milieu. These
changes were accompanied by alterations of ECaC traffick-
ing to or from the plasma membrane, respectively [36, 37].
Fig. 4. Effect of pendrin ablation on the expression of ECaC. (a). Northern blot analysis. ECaC mRNA expression decreased significantly in the kidney
cortices of pendrin KO mice. Our results demonstrate that the mRNA expression of ECaC decreased by ~58% in the cortex of Pds?/?compared to Pds1/1
mice (n ¼ 4 per genotype). (b). Immunoblot analysis. The protein abundance of ECaC decreased by 45% in the kidney cortices of Pds?/?mice.
1374 S. Barone et al.
The changes in cell surface expression of ECaC in acidic
pH environments were associated with congruent changes
in its activity [36, 37]. ECaC expression can also be altered
under systemic metabolic acidosis, which is associated
with the excretion of acidic urine . Under such condi-
tion, renal expression of ECaC decreases and there is a
concomitant increase in calcium excretion by the kidney
. Our studies demonstrated the downregulation of cal-
cium absorbing proteins both by northern and western blot-
ting in pendrin KO mice, suggesting that the acidic milieu
in the distal nephron lumen has additional effects on the
synthesis of these proteins.
Pds?/?mice do not display any evidence of systemic
metabolic acidosis [24, 27, 44]. Published reports indicate
that pendrin-null mice either demonstrate a normal acid-
base status or have a mild systemic metabolic alkalosis
subsequent to decreased bicarbonate excretion in the distal
nephron [24, 27, 44]. As such, we suggest that the down-
regulation of ECaC in pendrin-null mice is secondary to the
acidic luminal pH in the kidney distal nephron and is not
due to any systemic acid-base disturbances. The causal
relationship between the acidic urine and ECaC downregu-
lation was further supported by urine alkalinization studies,
which showed enhanced expression of ECaC in Pds?/?
mice subjected to oral bicarbonate loading (Results).
Oral bicarbonate loading causes urine alkalinization (Re-
sults) and results in metabolic alkalosis . We attempted
to examine the role of urine alkalinization independent of
metabolic alkalosis by subjecting the animals to bicarbonate
loading and ACTZ treatment. As shown, ACTZ-treated
Pds?/?mice displayed significantly elevated urine pH but
only a mild increase in their serum bicarbonate (Results),
consistent with urine alkalinization and the absence of meta-
bolic alkalosis. Urine calcium excretion was mildly reduced
in Pds?/?mice on oral bicarbonate and ACTZ (Figure 6c).
These results are not in conflict with our conclusion that
is a non-specific inhibitor of carbonic anhydrases and has
been shown to increase calcium excretion , presumably
secondary to the inhibition of sodium re-absorption in the
proximal tubule, which provides the driving force for cal-
cium re-absorption in that segment. In addition, ACTZ can
lower systemic pH, which may contribute to a rise in urinary
calcium excretion. We suggest that the calcium-reabsorbing
Fig. 5. Effect of pendrin ablation on the expression of Na/Ca exchanger and calbindin. (A) Na/Ca exchanger. (a) Northern blot analysis. The mRNA
levels of the basolateral Na/Ca exchanger decreased by ~54% in the kidneys of Pds?/?compared to Pds1/1mice. (b) Immunofluorescent labeling. Single
immunofluorescent labeling studies demonstrated significant reduction in the Na/Ca exchanger staining in Pds?/?(right panels) compared to Pds1/1
mice (left panels). (c) Immunoblot analysis. The protein abundance of the Na/Ca exchanger decreased by 41% in the kidneys of Pds?/?mice.
(B) Calbindin. (a) Northern hybridization. The mRNA levels of calbindin decreased by ~42% in the kidneys of Pds?/?compared to Pds1/1mice.
(b) Immunofluorescent labeling. Single immunofluorescent labeling studies suggested reduction in calbindin labeling in Pds?/?(right panels) compared
to Pds1/1mice (left panels). (c) Immunoblot analysis. The protein abundance of calbindin decreased by 32% in the kidneys of Pds?/?mice.
Pendrin, calcium excretion and kidney stone1375
effect of urine alkalinization in the distal tubule is likely
tubule in our Pds?/?mice on oral bicarbonate and ACTZ.
The present studies are the first reports on the impact of a
gene deletion causing low urinary pH with subsequent hy-
percalciuria. Our studies utilizing double immunofluores-
cence demonstrate that distal nephron bicarbonate secreting
cells (manifested by the expression of pendrin on their
apical domain) are distinct from calcium-absorbing cells
(as shown by Na/Ca exchanger localization on their baso-
lateral membrane) but both are present in the same tubule
(Figure 7). These results indicate that impaired bicarbonate
secretion will affect the luminal pH in tubules expressing
ECaC, suggesting that urine pH is working in a ‘paracrine’
fashion to regulate ECaC expression and or activity. A
recent study indicated that pendrin ablation leads to cal-
cium oxalate stone formation in the inner ear , lending
support to the possibility that ECaC regulation by extrac-
ellular pH (or bicarbonate) is observed in other organs.
It is plausible that the impact of urine alkalinization on
reducing the magnitude of hypercalciuria in pendrin KO
mice is secondary to several factors, one of which is the
upregulation of ECaC/calbindin/Na/Ca exchanger path-
way. Recent studies showed that bicarbonate loading in-
creased calcium absorption in the distal nephron in the
absence of ECaC, albeit at a lower rate .
Hypercalciuria, or increased calcium excretion, plays an
important role in the pathogenesis of various kidney stones,
such as calcium oxalate, which is the most common kidney
stone in humans, and calcium phosphate stones [46–50].
Patients with increased oxalate excretion might be in dan-
ger of developing kidney stones if the concentration of
calcium in their urine exceeds normal limits [46–50]. In a
recently published study, Worcester and Coe  found
that patients with calcium oxalate stones have acidification
defects in their kidney distal tubules.
The important role of urinary pH in the prevention of
kidney stones has been well documented in patients with
cystine stones who showed significant reduction in the for-
mation of cystine stones in response to oral alkalinization
(reviewed in ). In addition to its role in renal calcium
wasting, acidic urine can contribute to the formation of uric
Fig. 6. Effect of oral bicarbonate therapy on urine pH and calcium excretion. (a) Balanced studies in Pds1/1and Pds?/?mice. Body weight, food intake,
water intake and urine output were measured at baseline (top panel) and in response to oral bicarbonate loading (bottom panel). (b) Urine pH and calcium
excretion in Pds1/1and Pds?/?mice. As shown, urine pH was low in Pds?/?mice at baseline (bottom panel) and increased in both Pds1/1and Pds?/?
mice on oral bicarbonate therapy. Urinary calcium excretion (top panel) was higher in Pds?/?mice at baseline but decreased in response to oral
bicarbonatetherapy. (c). Urine pH andcalcium excretion in Pds?/?mice on ACTZand oralbicarbonate.As shown,urine pH increasedin Pds?/?miceon
oral bicarbonate therapy and ACTZ versus baseline conditions (left panel). Urinary calcium excretion decreased mildly in Pds?/?mice treated with
ACTZ and oral bicarbonate versus baseline conditions (right panel).
1376S. Barone et al.
acid stones in the kidneys, ureters or bladder if uric acid
excretion exceeds the normal limit [52, 53]. In convincing
studies by Pak et al. [52, 53], patients with idiopathic uric
acid nephrolithiasis, without secondary causes (such as de-
hydration or diarrhea), were found to have urinary pH val-
ues that were lower than control subjects, suggestive of a
primary defect in urinary acidification. While the patho-
genesis of acidic urine has not been studied in detail in
patients with primary uric acid stone, it is plausible that
alterations in acid-base transporters such as pendrin in the
distal nephron might contribute to this abnormality.
Based on its location and function, it is speculated that
pendrin mediates the post-prandial alkaline tide, a temporary
increase in urine pH immediately following a meal. The ‘al-
kaline tide’ is important to normal kidney function for the
elimination of ‘uric acid’ generated from meals  and is
lost in patients who make uric acid stones . It is intriguing
to speculate that pendrin-mediated bicarbonate secretion in
the distal nephron plays a pivotal role in the excretion of uric
acid by preventing its precipitation in the distal tubules.
In summary, deletion of pendrin downregulates the cal-
cium-absorbing pathway molecules (ECaC, calbindin and
Na/Ca exchanger) in the distal nephron and causes calcium
wasting. The downregulation of ECaC and related mole-
cules is likely secondary to the acidic urine resulting from
the inhibition of bicarbonate secretion in the distal nephron
in Pds?/?mice. Urine alkalinization reversed the impact
of pendrin deficiency on ECaC and calcium excretion by
enhancing the expression of ECaC and re-absorption of
calcium in the distal nephron.
We propose that acidic urine, as observed in pendrin KO
uric acid and possibly cystine stones. By causing acidic urine,
text of high urine oxalate or phosphate, will provide the right
milieu for calcium oxalate or calcium phosphate stone forma-
tion. In parallel, by causing acidic urine, pendrin ablation can
potentially facilitate the precipitation of uric acid (or cystine)
acid (or cystine) are enhanced. Future studies should investi-
gate whether single nucleotide polymorphisms in the pendrin
in humans with calcium or uric acid stones.
Acknowledgements. These studies were supported by a Merit Review grant
fromthe Departmentof Veterans Affairs and the National Institute of Health
Grant DK 62809 (to M.S) and by grants from US Renal Care (to M.S).
Conflict of interest statement. None declared.
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Received for publication: 19.5.11; Accepted in revised form: 26.7.11
Pendrin, calcium excretion and kidney stone1379