J Am Soc Nephrol 9: 2067-2074, 1998

Reduced Expression of the Renal Calcium-Sensing Receptor

in Rats with Experimental Chronic Renal Insufficiency

ROBERT S. MATHIAS,* HIEP T. NGUYEN,t MARTIN Y.H. ZHANG,* and

ANTHONY A. PORTALE*�Division of Pediatric Nephrology, Departments of *pediatrics, tPediatric Urology, and ‘�Medicine, tile

University of California San Francisco, San Francisco, California.

Abstract. Chronic renal insufficiency is associated with ebe-

vated serum parathyroid hormone (PTH) levels (2#{176}HPT), de-

ficiency of 1,25-dihydroxyvitamin D (l,25(OH)2D), and hy-

pocalciuria. In chronic renal insufficiency, the 2#{176}HPT may

result from reduced expression of the parathyroid gland extra-

cellular Ca2tsensing receptor (CaSR). Since the CaSR was

cloned from rat and human kidney, this study examined in rats

whether expression of the renal CaSR is altered in experimen-

tab chronic renal insufficiency. Four weeks after chronic renal

insufficiency was induced by 5/6 nephrectomy (Nx) in Sprague

Dawley rats, the serum creatinine concentration was 0.96 ±

0.06 mg/db compared with 0.35 ± 0.02 mg/dl in sham-operated

animals (P < 0.05). The serum total Ca2� and phosphorus

concentrations were not different. In the Nx group, the serum

concentration of aniino-PTH was higher (65 ± 8 pg/ml), and

the concentration of 1 ,25(OH)2D was significantly lower

(47 ± 5 pg/mb) compared with 45 ± 5 pg/mb and 6 1 ± 4 pg/mb

(P = 0.05) in the sham group, respectively. In a subset of rats

studied, the Nx group was hypocalciuric (1 .4 ± 0.5 mg/kg per

d) compared with the sham group (3.7 ± 0.5 mg/kg per d) (P <

0.05). In the Nx rats, CaSR mRNA expression and CaSR

protein levels were found to be reduced by 35 and 38%,

respectively, than those observed in controls. These results

suggest that reduced renal CaSR expression in chronic renal

insufficiency may play a role in disordered mineral ion ho-

meostasis, including hypocalciuria.

The G protein-coupled extraceblubar calcium (Ca2�)-sensing

receptor (CaSR), recently cloned from bovine parathyroid

gland (1) and rat and human kidney (2,3), provides a mecha-

nism by which changes in the extracellular Ca2� concentration

([Ca210) can regulate, either indirectly or directly, mineral ion

homeostasis. A small decrease in [Ca210 is sensed by the

parathyroid cell, which increases its secretion of parathyroid

hormone (PTH) (4). PTH acts both on the skeleton to increase

bone resorption, and on the kidney to increase the tubular

reabsorption of Ca2� and the synthesis of 1 ,25-dihydroxyvita-

mm D (l,25(OH)2D), thus promoting an increase in [Ca2�j0 to

normal levels. In the kidney, changes in [Ca2�]0 can directly

modulate the synthesis of l,25(OH)2D (5), the expression of

vitamin D-dependent calbindin-D2SK (6), and the tubular reab-

sorption of Ca2� in the thick ascending limb of the loop of

Henle (7,8). The finding that the CaSR has been localized to

segments of the proximal and distal nephron (9) provides a

mechanism by which a direct effect of [Ca2�]0 can modulate

renal tubule function and, thereby, mineral ion homeostasis, at

beast in part, via receptor-mediated events.

Normal expression of the CaSR can be essential in main-

taming mineral ion homeostasis. In patients with familial hy-

Received December 4, 1997. Accepted May 4, 1998.Correspondence to Dr. Robert S. Mathias, Children’s Renal Center, University

of California, San Francisco Medical Center. 533 Parnassus Avenue, RoomU-585, San Francisco, CA 94143-0748.

1046-6673/0901 l-2067$03.00/0

Journal of the American Society of Nephrology

Copyright © 1998 by the American Society of Nephrology

pocabciuric hypercalcemia (FHH) and neonatal hyperparathy-

roidism, inactivating mutations in the CaSR gene have been

identified (10). In such patients, mutations of the CaSR give

rise to disordered mineral-ion homeostasis, specifically, hyper-

parathyroidism, hypocalciuria, and hypercalcemia. In addition,

these symptoms can be induced in mice by knocking out the

CaSR gene (1 1).

With progressive chronic renal insufficiency, disturbances in

mineral ion homeostasis, including secondary hyperparathy-

roidism (2#{176}HPT) (12), play an important role in the pathogen-

esis of metabolic bone disease. Hypocalciuria also is observed

in patients with mild and moderate chronic renal insufficiency

(13,14). In chronic renal insufficiency, there is evidence that

these disturbances reflect, at least in part, an abnormality in the

function of the CaSR. Indeed, expression of the CaSR mRNA

and its protein were reduced in hyperplastic parathyroid glands

from hemodiabysis patients with severe chronic 2#{176}HPT when

compared with that in normal glands from patients with para-

thyroid adenomas (15,16).

In patients with FHH, both the hypocalciuria and the abnor-

mality in [Ca2�i0-regulated PTH secretion, specifically, the

increase in the Ca2� set-point, are thought to reflect abnormal

function of the CaSR (17). Accordingly, as occurs in patients

with FHH and in mice lacking the CaSR gene in which urinary

Ca2� excretion is greatly decreased, a reduction in expression

of the CaSR in the kidney might contribute to the hypocalciuria

observed in patients with chronic renal insufficiency (13,14).

To test this hypothesis, we determined whether renal expres-

sion of the CaSR and urinary calcium excretion were reduced

in the rat with moderate chronic renal insufficiency.

2068 Journal of the American Society of Nephrology J Am Soc Nephrol 9: 2067-2074, 1998

Materials and MethodsAnimals and Diets

Sprague Dawley rats weighing 200 to 250 g were fed normal rat

chow (Purina) throughout the study and were allowed water ad

libitum. The diet contained 0.93% calcium and 0.81% phosphorus.

Rats underwent either sham surgery or one-stage 5/6 nephrectomy

(Nx) ( 18, 19). Briefly, rats were anesthetized with Nembutal, and an

incision was made through the skin and body wall along the midline.

The left kidney was exposed, decapsulated, and in rats subjected to5/6 Nx, the upper and lower branches of renal arteries were ligated.

The right kidney was then exposed, decapsulated, and in rats sub-

jected to 5/6 Nx, the renal pedicle was ligated and the kidney was

removed. The incision was then closed. Four weeks after surgery,

urine was obtained for a 24-h period for the determination of creati-

nine (Cr) and total Ca2� (TCa2� ) concentration. At the time of

sacrifice, blood was obtained for determination of serum concentra-

tions of Cr, TCa2�, phosphorus (P), PTH, and l,25(OH),D, and the

kidneys were snap-frozen in liquid nitrogen. The procedures were

approved by the Committee on Animal Research of the University of

California, San Francisco.

Biochemical Determinations

Plasma (TCa2�, P, Cr) and urine (Ca2�, Cr) concentrations were

determined by an autoanalyzer (model 747; Boehringer Mannheim,

Indianapolis, IN). Serum immunoreactive N-terminal PTH (iPTH)

was determined in duplicate with a rat PTH (immunoradiometric

assay) kit (Nichols Institute Diagnostics, San Juan Capistrano, CA).

Serum levels of l,25(OH)2D were determined in duplicate by radio-

receptor assay (Nichols Institute Diagnostics).

Isolation of Total RNA

Total tissue RNA was extracted by homogenizing kidney tissue

with a glass tissue grinder in RNA STAT-60 (Tel-Test “B”, Inc.,

Friendswood, TX) as a modification of the guanidinium isothiocya-

nate method (20). The RNA concentration was determined by absor-

bance at 260 nm.

Ribonuclease Protection Analysis

The RNase protection assay was performed as described (Hyb-

Speed RPA assay kit, Ambion, Austin, TX). A l272-bp (PstI-SacI)

fragment of the rat kidney CaSR cDNA (NPS Pharmaceuticals, Salt

Lake City, UT) (2) was subcloned into pBlueScript SK+ immediately

downstream from the bacteriophage T3 promoter. The rat kidney

CaSR fragment consists of the coding region sequence starting at

transmembrane region 4 and extending beyond the C-terminal stop

codon (from exon 7 of the CaR gene). A 250-bp fragment of theglyceraldehyde phosphate dehydrogenase (GAPDH) eDNA was sub-

cloned into pT7Blue(R) immediately downstream from the T7 pro-

moter. The CaSR and OAPDH eDNA were linearized with ApaI

(Promega, Madison, WI) and HphI (New England Biolabs, Beverly,

MA), respectively. The radiolabeled antisense RNA probes weretranscribed separately from the linearized plasmids using T3 polymer-

ase and T7 polymerase, respectively (Maxiscript protocol, Ambion),

in the presence of [a-32P]-labeled UTP (New England Nuclear, Bos-

ton, MA). The predicted sizes of the protected fragments for the CaSR

and GAPDH were 336 bp and I 64 bp, respectively. The labeled

probes (2 p.1; 5 X b0� cpm) were placed together with sample total

RNA and hybridized for 20 mm at 68#{176}C.After hybridization, the

mixture was treated with ribonuclease at 37#{176}Cfor 30 mm to degrade

single-stranded RNA and unhybridized probe. The remaining pro-

tected RNA fragments were precipitated and dried. Each pellet was

dissolved in loading buffer (Ambion), denatured at 90#{176}C,and resolved

on a 5% acrylamide!8 M urea sequencing gel. The gel was placed on

x-ray film (Hyper-fllm MP, Amersham, Arlington Heights, IL) with

an intensifying screen and exposed at -80#{176}C for I to 5 d for

autoradiography. The intensity of the CaSR mRNA was visualized

and normalized to that of GAPDH mRNA using densitometric values

obtained by National Institutes of Health Image I .60 software (Be-

thesda, MD). Results are expressed as the ratio CaSR/GAPDH

mRNA. Total RNA from bovine parathyroid glands was kindly sup-

plied by Dr. Dolores Shoback (Veteran’s Administration Medical

Center, San Francisco, CA).

Protein Preparation

Crude membranes from kidney tissue were prepared by a modifi-

cation of the method by Chattopadhyay et al. (2 1). Briefly, 250 mg of

kidney tissue was homogenized in buffer: ice-cold 50 mM Tris-HC1,

pH 7.4, 0.30 M sucrose, 1 mM ethyleneglycol-bis-(�-aminoethylether)-N,N,N’,N’-tetra-acetic acid, ethylenediamine tetra-acetic acid,

and protease inhibitors (80 p�g/ml aprotinin, 30 p.g/ml leupeptin, I

mg/ml Pefabloc SC, 50 p.g!ml calpain inhibitor, 50 p.g/ml bestatin,

and 5 �tg/ml pepstatin). Nuclei and cell debris were removed from the

resultant homogenate by centrifugation (12,000 X g) for 20 mm at

4#{176}C.The supernatant was centrifuged at 42,000 X g for 20 mm at

4#{176}C,and the remaining pellet, representing a crude membrane prep-

aration, was solubilized with 1% Triton X-lOO in 100 p.1 of lysis

buffer and mixed with 2X Laemmli sample buffer containing 1 mM

dithiothreitol. Cultured human embryonic kidney-293 cells (HEK-

CaSR) stably expressing the human parathyroid Ca2�-sensing recep-

tor (NPS Pharmaceuticals) were grown to confluence and rinsed with

phosphate-buffered saline. Cells were scraped, then pelleted at 3500

rpm for 3 mm at 4#{176}C.Supernatant was removed, and lysis buffer

containing I X Hepes-buffered saline, pH 7.4, 1% Triton X-bOO, and

protease inhibitors (10 p.g/ml each ofaprotinin, leupeptin, and calpain

inhibitor) was added to the pellet. Protein concentrations were deter-

mined by the BCA protein reagent kit (Pierce, Rockford, IL).

Western Blot Analysis

Proteins obtained from HEK-CaSR cells and kidney from sham-

operated and 5!6 Nx rats were separated by 6% sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (PAGE) (22) and trans-

ferred to nitrocellubose filters (Amersham) electrophoretically at 0.2

mV for 3 h in transfer buffer contain 3 mM Tris-base, 190 mM

glycine, 0.01% sodium dodecyl sulfate, and 20% methanol. The filters

were blocked with 5% milk containing 20 mM Tris-base, pH 7.6, 137

mM NaC1, and 0. 1 % Tween-20 (TBS-T) overnight, then washed 3

times in 0.1% milk and one time in 1% milk in TBS-T (5 mm each)

at room temperature. The filters were then incubated with protein

A-purified, anti-CaSR antibody 4641 (1:10,000 dilution) in 1% milk

containing TBS-T for 1 h at room temperature. This anti-CaSR

primary antibody was raised to a peptide corresponding to the de-

duced sequence of amino acids 2 15 to 237 (DDDYORPGIEKFREE-

AEERDIC) of the extracellular domain of the bovine parathyroidCaSR (1), which has a sequence identical to that of the rat kidney

CaSR (2). The filters were washed 3 times in 0. 1% milk and one time

in 1% milk in TBS-T (5 mm each) at room temperature. The second-

ary antibody (horseradish peroxidase-coupled anti-rabbit IgO; Amer-

sham) in 1% milk and TBS-T (1:10,000 dilution) was incubated with

the filters for 1 h at room temperature. In a similar sequence, the filters

were washed with milk-containing solutions as before. The specific

bands were visualized using enhanced chemiluminescence (ECL)

detection (Amersham) after exposing the filter to film (Hyperfilm-

J Am Soc Nephrol 9: 2067-2074. 1998 Reduced Renal CaSR in Renal Insufficiency 2069

ECL, Amersham). Results were expressed as arbitrary densitometric

units per milligram of protein added to each lane, using National

Institutes of Health Image 1 .60 software (Bethesda, MD). Crude

membrane protein from HEK-CaSR cells was used as a positive

control.

Statistical AnalysisAll data are expressed as means ± SEM. Statistical analysis was

performed by unpaired t test, using StatView for the Macintosh. A P

value of �0.05 was taken to indicate a statistically significant differ-

ence.

ResultsBiochemical Results

Four weeks after surgery, the body weights of the 5/6 Nx

animals were significantly lower than those of sham-operated

animals (P < 0.05) (Table 1). The serum Cr concentrations in

the Nx animals were higher than those in sham animals (P <

0.05). The serum concentrations of TCa2� and P were not

significantly different between the two groups. The concentra-

tions of N-terminal PTH in the Nx group were higher and of

l,25(OH)2D were significantly lower (P = 0.05) than those in

the sham-operated animals.

Because decreased urinary Ca2� excretion has been noted in

patients with mild-to-moderate chronic renal insufficiency

( 13, 14), we determined whether urinary Ca2� excretion was

decreased in experimental chronic renal insufficiency in the

rat. After 4 wk of surgery, nine animals from each group were

placed in metabolic cages. After a 48-h acclimation period,

urine was collected over a 24-h period for determination of

Ca2� and Cr (Table 2). Urinary excretion of Ca2� and the

creatinine clearance rate (Car) were significantly lower in the

Nx animals than in the sham-operated animals (P < 0.05). In

this subset of animals, the serum bevels of TCa2�, P, and iPTH

Table 1. Body weights and biochemical determinations

obtained at the time of sacrifice in sham-operated

and 5/6 Nx ratsa

Characteristic Sham 5/6 NxValue

n 15 19

Body weight (g)

presurgery 237 ± 3 236 ± 3 NS

at sacrifice 346 ± 4 316 ± 7 <0.05

Serum

Cr (mg/dl) 0.35 ± 0.02 0.96 ± 0.06 <0.05total Ca2� (mg/db) 9.9 ± 0.2 10.0 ± 0.1 NS

P (mg/dl) 9.2 ± 0.2 9.7 ± 0.2 NS

iPTH (pg/mI) 45 ± 5b 65 ± 8C 0.06

l,25(OH)2D (pg/ml) 61 ± 4b 0.05

a Results are expressed as means ± SEM. Cr, creatinine; P,

phosphorus; iPTH, immunoreactive N-terminal parathyroid

hormone; l,25(OH),D, 1,25-dihydroxyvitamin D.C, Represents 14 animals.

C Represents 16 animals.

Table 2. Biochemical determinations obtained at the time of

sacrifice in sham-operated and 5/6 Nx ratsa

..

Charactenstic Sham 5/6 NxP

Value

n 9 9

Serum

total Ca2� (mg/db) 9.9 ± 0.1 10.2 ± 0.1 <0.05

P (mg/dl) 9.3 ± 0.2 9.7 ± 0.2 NS

iPTH (pg/mb) 53 ± 5 72 ± 6 <0.05

l,25(OH)2D (pg/ml) 46 ± 3 33 ± 4 <0.05

Urine’�

C�r (mb/mm per kg) 10.2 ± 0.6 1.9 ± 0.4 <0.05

24-h urine Ca2� (mg/kg) 3.7 ± 0.5 1 .4 ± 0.5 <0.05

a All values are the mean ± SEM. C�r creatinine clearance rate.

Other abbreviations as in Table I.

b Calculated using body weight at time of sacrifice.

(P < 0.05) were higher, and those of 1 ,25(OH)2D (P < 0.05)

were lower in the Nx group than in the sham-operated group.

Ribonuclease Protection Analysis

To determine CaSR mRNA expression in rat kidney and in

bovine parathyroid tissue, RNase protection assay was per-

formed using total RNA prepared from normal kidney and

bovine parathyroid tissue (Figure 1). A protected fragment of

the expected size for the CaSR was observed in rat kidney and

bovine parathyroid tissue, but not in yeast and untransfected

HEK cells (Figure 1). Total RNA from yeast and untransfected

Kid PTG Yst HEK

CaSR �

bp

�- 336

GAPDH � � ..l�- 164

Figure 1. Expression of extracellular calcium (Ca2 � )-sensing receptor

(CaSR) mRNA in rat kidney and bovine parathyroid gland tissue asdetermined by RNase protection assay. Total RNA (10 p.g per lane)

was prepared and hybridized with the radiolabeled antisense RNA

probes as described in Materials and Methods for the following

tissues: normal rat kidney (Kid), bovine parathyroid gland (PTO),

yeast (Yst), and untransfected human embryonic kidney (HEK) cells.

The expected size of the protected RNA fragments was 336 bp for theCaSR and I 64 bp for glyceraldehyde phosphate dehydrogenase

(OAPDH).

.� 1

C-.

� 1

�

� 0.5

C-.) 0.25

p<0.05

2070 Journal of the American Society of Nephrology J Am Soc Nephrol 9: 2067-2074, 1998

Nx

,,,,‘ ,

Sham

CaSR

GAPDH

Figure 2. The effect of chronic renal insufficiency on CaSR mRNA expression in rat kidney as determined by RNase protection assay. Total

RNA was obtained from the remnant kidney of 5/6 nephrectomized (Nx) rats and from both of the kidneys of sham-operated (Sham) animals

at 4 wk after completion of the surgery. RNA (20 p.g per lane) was hybridized with the radiolabeled antisense RNA probes as described in

Materials and Methods. Lanes 1 through 9 represent RNA obtained from 5/6 Nx animals, and lanes 10 through I 8 represent RNA obtained from

sham animals.

HEK cells were used as negative controls. The expression of

GAPDH mRNA was used as an internal reference standard.

To determine whether expression of the kidney CaSR

mRNA was altered by 4 wk of chronic renal insufficiency of

moderate severity, RNase protection assay was performed us-

ing total RNA prepared from kidneys of 5/6 Nx and sham-

operated animals 4 wk after surgery. To control for the possi-

bility that differences in CaSR mRNA expression were due to

differences in RNA loading, the expression of GAPDH mRNA

was also determined. As shown in a representative autoradio-

graph, CaSR mRNA expression was lower in kidney of 5/6 Nx

animals than in sham-operated animals (Figure 2). Densitomet-

nc quantification of CaSR mRNA expression from three dif-

ferent experiments, normalized to that of GAPDH mRNA

expression, was 35% lower in the 5/6 Nx animals than in

sham-operated animals (P < 0.05) (Figure 3). Thus, in the rat

with chronic renal insufficiency, expression of CaSR mRNA

was reduced substantially. GAPDH mRNA expression was

observed to be unaffected by 5/6 Nx (Figure 2) when an equal

amount of total RNA from either the sham-operated or 5/6 Nx

group was loaded in each lane during each experiment.

Western Blot Analysis

To examine the expression of CaSR protein in the kidney,

Western blot analysis was performed on membrane proteins

harvested from kidney tissue. We first determined whether the

anti-CaSR antibody 4641 was specific for CaSR protein by

harvesting proteins from HEK-293 cells stably expressing the

human parathyroid CaSR cDNA (HEK-CaSR) and from nor-

mal rat kidney. The CaSR-specific antibody 4641 detected

CaSR protein in both HEK-CaSR cells and normal rat kidney

(Figure 4), but not in untransfected HEK cells (not shown). In

HEK-CaSR cells, the specific staining pattern for the CaSR

protein, using the anti-CaSR antibody, demonstrates a doublet

of immunoreactive bands with molecular weights between 1 17

and 206 kD, similar to those observed for the glycosylated

receptor products (approximately 140 to 150 and approxi-

mately 160 to 170 kD) (2 1 ). In kidney, the anti-CaSR antibody

demonstrates a single broad band or a doublet with molecular

weights between 1 17 and 140 kD, which represents the glyco-

sylated receptor; the band observed in our experiments below

1 17 kD represents a degradation product of the CaSR protein

(23). We also observed a high molecular weight band above

206 kD, which is thought to represent dimers of the receptor

(24).

We next determined whether the expression of the CaSR

protein was reduced in 5/6 Nx animals compared with sham-

operated animals. A representative autoradiograph of crude

kidney membrane protein from 5/6 Nx and sham-operated

animals was analyzed for CaSR expression by Western blot

(Figure 5). Densitometric quantification of the single broad

band between 1 17 and 140 kD from autoradiographs from

three different experiments demonstrates that the expression of

CaSR protein was 38% lower in Nx animals than in sham-

operated animals (Figure 6). This broad band was chosen

because the predicted size of the CaSR membrane protein is

approximately 120 kD (1). Thus, these data demonstrate that

both CaSR mRNA expression and CaSR protein content are

reduced by experimental chronic renal insufficiency in the rat.

Sham 5/6Nx

Figure 3. Densitometric quantification of kidney CaSR mRNA ex-

pression normalized to OAPDH mRNA expression 4 wk after surgery

from 15 sham and 19 Nx rats. Data are given as mean ± SEM.

J Am Soc Nephrol 9: 2067-2074, 1998 Reduced Renal CaSR in Renal Insufficiency 2071

HEK

CaSR Kidney

MW(kDa)

-�-2O6

-Is CaSR�-117

Figure 4. Expression of CaSR protein in HEK-293 cells stably trans-

fected with the human parathyroid CaSR (HEK-CaSR) and normal rat

kidney as determined by Western blot analysis. Membrane proteins

were obtained from HEK-CaR cells and normal rat kidney. Membrane

protein was added to each lane, subjected to polyacrylamide gel

electrophoresis (PAGE), electrotransferred to nitrocellulose filters,

and blotted using CaSR-specific antiserum 4641 (see Materials and

Methods). Lane I represents 4-p.g membrane protein obtained from

HEK-CaSR cells, and lanes 2 and 3 represent 50-p.g membraneprotein obtained from normal rat kidney. For both HEK-CaSR cells

and kidney, the broad specific bands above 206 kD represent dimers

of the receptor (24). The distinct doublet of immunoreactive bands

(HEK-CaR cells) and the single broad band or doublet (rat kidneytissue) between the 1 17- and 206-kD markers are compatible with the

glycosylated CaSR translated in vitro ( 1 ). The band that is detected

below I 17 kD in rat tissue represents a degradation product of the

CaSR protein (23).

DiscussionA CaSR from human and rat kidney was recently cloned

(2,3), and its predicted amino acid sequence shares approxi-

mateby 92% identity with that of the CaSR from bovine para-

thyroid glands (2). The identification of a CaSR in the kidney

provides a mechanism by which changes in [Ca2�]() can mod-

ulate renal tubular function directly (5-8). For example, high

[Ca2110 can inhibit both PTH-dependent cAMP production in

renal cortical cells (25) and vasopressin-dependent cAMP pro-

duction in the thick ascending limb of the loop of Henle (26).

These observations suggest that the CaSR plays an important

role in the regulation of renal tubular function.

In the present study, we examined the expression of the renal

CaSR in experimental moderate chronic renal insufficiency in

the rat. In rats with 5/6 Nx, the estimated GFR was reduced by

approximately 60%, serum PTH levels were increased, and

both serum l,25(OH)2D levels and urinary Ca2� excretion

were decreased when compared with sham-operated rats. We

found that the expression of the CaSR mRNA, as determined

by RNase protection assay, was decreased by 35% in 5/6 Nx

animals when compared with that in sham-operated animals.

We also found that the CaSR protein content, determined by

Western analysis, was reduced by 38%.

We calculated CaSR mRNA expression as the ratio of CaSR

mRNA to GAPDH mRNA. It is possible that in the hypertro-

phic remnant kidney of Nx animals, expression of the house-

keeping gene GAPDH is altered. In our studies, however, we

found no obvious changes in GAPDH mRNA expression in 5/6

Nx animals when compared with sham-operated animals. Sim-

ilarly, in studies of rats in which the degree of experimental

chronic renal insufficiency was similar to that in the present

study, GAPDH mRNA expression in both the kidney (27) and

liver (28) was unaffected when compared with the sham group.

Furthermore, the renal expression of 285 ribosomal RNA and

j3-actin mRNA was unaffected by chronic renal insufficiency

in the rat (29). Thus, our findings of reduced CaSR mRNA

relative to that of GAPDH mRNA in the kidney cannot be

attributed to altered expression of the housekeeping gene

GAPDH.

When we performed Western analysis, CaSR proteins from

kidney were detected at slightly lower molecular weights than

those obtained from HEK cells expressing the human parathy-

roid CaSR. This finding suggests that the protein expressed by

cells transfected with the parathyroid CaSR is different from

that expressed natively in renal tubular cells. Such a difference

might reflect differences in processing of the CaSR cDNA in

transfected cells when compared with renal tubular cells. A

second possibility is that there may be differences in posttrans-

lational modification of the protein. An alternative possibility

is that the rat kidney expresses different isoforms of the CaSR

than does human parathyroid tissue.

Little is known about the regulation of CaSR gene expres-

sion. In rats with either vitamin D deficiency (30,3 1 ), low

dietary Ca2� (32), or chronic renal insufficiency induced by

5/6 Nx (32) (three conditions in which serum PTH levels are

increased and [Ca2�]() is decreased), parathyroid gland CaSR

mRNA expression was not significantly different from control

values. Moreover, serum 1 ,25(OH),D levels were either de-

creased (30,3 1 ) or increased (32), suggesting that I ,25(OH),D

had no effect on parathyroid gland CaSR mRNA. In contrast,

Brown et a!. demonstrated that in rats fed a vitamin D-deficient

diet, CaSR mRNA expression in both the parathyroid gland

and kidney was decreased (3 1 ), and that administration of

l,25(OH),D3 resulted in an increase in CaSR mRNA levels to

near normal (3 1 ). Thus, in the present study, the finding of

decreased CaSR expression in kidney from rats with chronic

renal insufficiency might be attributed at least in part to the

observed decrease in their serum concentrations of

1 ,25(OH),D.

The 2#{176}HPTassociated with severe chronic renal insuffi-

ciency is thought to result not only from reduced serum levels

of 1 ,25(OH),D, but also from reduced sensitivity of the para-

thyroid gland to the suppressive effect of increased [Ca2�]() on

PTH secretion (1 2). This latter mechanism was first suggested

by Brown et a!. , who demonstrated that the concentration of

[Ca2�]0 required to suppress PTH secretion in vitro was higherin hyperplastic parathyroid glands from uremic individuals

with 2#{176}HPT than in normal glands (33). This notion was

further supported by observations that in hemodialysis patients

with 2#{176}HPT,the Ca2� set-point was increased in vivo (34). It

10

8

6

4

2

U)

.� !�

UU)C.)

�0

p<0.05

2072 Journal of the American Society of Nephrology J Am Soc Nephrol 9: 2067-2074, 1998

Nx Sham

‘: �-

Figure 5. The effect of chronic renal insufficiency on CaSR protein expression in rat kidney as determined by Western blot analysis. Membrane

proteins were obtained from the kidney remnant of 5/6 Nx rats and from both kidneys of sham-operated animals 4 wk after surgery. A total

of 50 i.�g of membrane protein was added to each lane, subjected to PAGE, electrotransferred to nitrocellulose filters, and Western blots were

performed using CaSR-specific antiserum 4641 (see Materials and Methods). Lanes 1 through 5 represent protein obtained from 5/6 Nx

animals, and lanes 6 through 9 represent protein obtained from sham-operated (Sham) animals.

Sham 5/6 Nx

Figure 6. Densitometric quantification of kidney protein expression 4

wk after surgery in 15 sham and 18 nephrectomized animals. Results

are shown as mean ± SEM.

should be noted, however, that in some dialysis patients with0 � . �±2 HPT, no increase in the Ca set-point has been detected

(35). Nevertheless, it has been suggested that such abnormal

Ca2� sensing by the parathyroid gland in chronic renal insuf-

ficiency reflects an abnormality in function of the parathyroid

cell CaSR. In fact, the expression of the CaSR was reduced in

parathyroid cells from patients undergoing chronic dialysis

when compared with that in parathyroid tissue from healthy

subjects (15,16).

In contrast to the severe chronic 2#{176}HPTfound in patients

with chronic renal failure undergoing long-term hemodialysis,

the biochemical disturbances observed in our study of 5/6 Nx

animals more likely represent mild-to-moderate 2#{176}HPT and

moderate renal insufficiency (Table 2). Such early 2#{176}HPT

could be the result of decreased serum bevels of 1 ,25(OH)2D.

However, we found that CaSR expression is reduced, at beast in

the kidney, and potentially in the parathyroid gland, early in the

development of renal insufficiency, and suggest that such

changes may play an important role in the early development

of disturbed mineral ion homeostasis, i.e. , 2#{176}HPTand possibly

l,25(OH)2D deficiency as well.

In patients with mild-to-moderate chronic renal insuffi-

ciency, urinary Ca2� excretion is significantly lower than that

in subjects with normal renal function (13,14). In our study, we

also found that urinary Ca2� excretion was reduced in animals

with experimental chronic renal insufficiency when compared

with sham-operated controls. In patients with FHH who harborinactivating mutations in the CaSR gene (10) and in mice in

which the CaSR gene has been knocked out (1 1), both exhibit

hypocalciuria. Furthermore, in patients with FHH rendered

hypoparathyroid by total parathyroidectomy, the reduced un-

nary Ca2� excretion persisted, providing evidence that dys-

function of the renal CaSR can mediate hypocalciunia, inde-

pendent of serum PTH bevels (36). Therefore, the observed

hypocalciuria in our study might also reflect an abnormality in

the function of the CaSR in the kidney that results from a

decrease in its renal expression.

An additional potential mechanism for the observed hy-

pocalciuria could reflect the stimubatory effect of increased

serum levels ofPTH on renal Ca2� reabsorption, but could also

reflect either deficiency of 1 ,25(OH)2D or resistance to its

actions on intestine and bone (14). However, the ability of PTH

to reduce urinary Ca2� excretion might be mitigated in patients

with chronic renal insufficiency because of resistance to the

actions of PTH in target tissues, such as bone (37,38), presum-

ably mediated by reduced expression of PTH receptors in such

tissues. Indeed, in 5/6 Nx rats, expression of PTH-PTH-releas-

ing protein (PTHrP) receptor mRNA was reduced in both

hepatocytes (28) and kidney (29), the batter tissue demonstrat-

ing marked reduction in PTH-dependent adenylate cyclase

activity (29). In patients with mild chronic renal insufficiency

and hypocalciuria, serum concentrations of both Ca2� and

1 ,25(OH)2D are reported to be not different from those in

controls (14). The observation that administration of

1 ,25(OH)2D3 to these patients induced an increase in urinary

Ca2� excretion (14) suggests that l,25(OH)2D3 resistance is

present in target tissues (i.e. , intestine and possibly bone) in

patients with mild-to-moderate chronic renal insufficiency.

Furthermore, the administration of 1 ,25(OH)2D3 to vitamin

D-deficient rats increased renal CaSR expression to near nor-

mal levels (3 1). Thus, another potential explanation for the

observed increased urinary Ca2� excretion in patients with

J Am Soc Nephrol 9: 2067-2074, 1998 Reduced Renal CaSR in Renal Insufficiency 2073

mild-to-moderate chronic renal insufficiency treated with

1 ,25(OH)2D3 is that this hormone resulted in increased renal

CaSR expression, and thereby improved renal tubular handlingof Ca2� . It remains to be determined whether administration of

1 ,25(OH)2D3 to animals with experimental chronic renal in-

sufficiency can alter the expression of the CaSR. Finally,

because we did not measure the urinary Ca2� ultrafiltrate, we

cannot determine whether the hypocalciuria observed in the 5/6

Nx animals was in part due to a decrease in the filtered load of

Ca2� with no change in its fractional reabsorption.

Decreased expression of calciotrophic hormone receptors in

the kidney and in other tissues could contribute to disorders of

mineral homeostasis in chronic renal insufficiency. For exam-

ple, reduced expression of PTH-PTHrP receptors in the kidney

could result in decreased phosphaturic effect of PTH on the

renal tubule and thereby contribute to the development of

hyperphosphatemia in chronic renal insufficiency. Similarly, in

experimental chronic renal insufficiency in the rat, the ob-

served resistance to the actions of growth hormone is due, at

least in part, to reduced expression of hepatic receptors for

insulin-like growth factor-b (39) and growth hormone (40).

These observations raise the fundamental question of the mo-

lecular basis for decreased receptor expression that is believed

to play an important role in the resistance to hormonal actions

in chronic renal insufficiency. Massry et al. observed that basal

levels of intracellular Ca2� were increased in hepatocytes and

cardiomyocytes from rats with experimental chronic renal in-

sufficiency, and hypothesized that such an increase of intra-

cellular Ca2� may induce a decrease in mRNA levels for

PTHrP receptors, and therefore be responsible for decreased

receptor expression in chronic renal insufficiency (28). More

recently, Baudouin-Legros et al. demonstrated that urea-in-

duced inhibition of cAMP production was reversed by protein

kinase inhibitors, thus suggesting that urea may directly inter-

fere with specific signaling pathways by altering cellular pro-

tein phosphorylation (41).

In summary, we demonstrate reduced expression of the

CaSR at the level of mRNA expression and protein content in

the kidney of rats with experimental chronic renal insuffi-

ciency. We hypothesize that reduced expression of the CaSR

gives rise to abnormal Ca2� sensing by the renal tubule, which

results in disordered tubular handling of Ca2�, and thereby

contributes to the hypocalciuria observed in patients with

chronic renal insufficiency. It would be of interest to perform

immunohistochemical localization of the CaSR in kidney from

animals with chronic renal insufficiency to determine whether

altered CaSR expression is specific in tubular segments along

the nephron, which may play a role in the development of

hypocalciuria found in chronic renal insufficiency.

AcknowledgmentsThis work was supported by National Institutes of Health Grant

MO1RRO127I, Pediatric Clinical Research Center, and by a grant

from the Oenentech Foundation. We are grateful to Drs. Forrest

Fuller, Jim Garrett, and Edward Nemeth (NPS Pharmaceuticals, SaltLake City, UT) for the kind gift of the CaSR riboprobe and anti-CaSR

antibody 4641. We also thank Dr. Edward Brown for his continued

support, suggestions, and discussions.

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