1 Nephrology and Hypertension Services, Hadassah University Hospital, Jerusalem, Israel 91120; and 2 Nephrology Section, The University of Texas Southwestern Medical Center and Dallas Veterans Affairs Medical Center, Dallas, Texas 75216
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Acute administration of dihydroxycholecalciferol [1,25(OH)2D3] blunts phosphaturia and increases urinary cAMP excretion in parathyroid hormone (PTH)-infused parathyroidectomized (PTX) rats. Because chronic administration of 1,25(OH)2D3 enhances the phosphaturic response to exogenous parathyroid hormone despite blunting of urinary cAMP excretion, we have examined the expression of the renal cortex type II Na-Pi cotransporter (NaPi-2) mRNA and protein in 1) chronic PTX Sabra rats, 2) PTX rats receiving a physiological dose of 1,25(OH)-2-D3, 3) PTX rats receiving 35 ng/h of PTH, and 4) rats receiving both PTH and 1,25(OH)2D3, for 7 days via osmotic minipumps. Our results confirm that there is increased phosphaturia in the PTH+1,25(OH)2D3-infused animals despite blunting of urinary cAMP excretion, a reduced filtered load of phosphate, and lack of a phosphaturic effect by 1,25(OH)2D3 alone. Both PTH and 1,25(OH)2D3 significantly reduced expression of renal cortex NaPi-2 mRNA and NaPi-2 protein, and the administration of PTH together with 1,25(OH)2D3 had additive effects in further decreasing NaPi-2 mRNA and NaPi-2 protein levels. Expression of two other epithelial transporters, type 1 Na-sulfate and type 1 Na-glucose cotransporters, were not different between the groups, suggesting specificity of the effects of PTH and 1,25(OH)2D3 on phosphate transport. The effect of chronic administration of 1,25(OH)2D3 has not been noted previously, and the cellular mechanisms and signaling processes that mediate the decrease in NaPi-2 remain to be determined.
type II sodium-phosphate cotransporter; parathyroid hormone; parathyroid hormone receptor; dihydroxycholecalciferol; phosphaturia
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
WE HAVE PREVIOUSLY REPORTED that the acute phosphaturic effect of parathyroid hormone (PTH) is blunted by 25 hydroxycholecalciferol [25(OH)D3], 24,25 dihydroxycholecalciferol [24,25(OH)2D3], and 1,25 dihydroxycholecalciferol [1,25(OH)2D3] (2, 4, 17). This response is associated with decreased urinary excretion of cAMP and reduced PTH-induced in vitro activation of adenylate cyclase in the renal cortex. The mechanism of this acute action of vitamin D metabolites is unclear but appears to be dependent on posttranscriptional protein synthesis (2), independent of cytoplasmic microtubule integrity (5), and probably calmodulin dependent (3). Because dibutyryl cAMP infusion-induced phosphaturia is not decreased by vitamin D metabolites (17) and these metabolites exert no acute antiphosphaturic effect when administered alone, it appears that vitamin D metabolites inhibit hormone-induced phosphaturia by direct interference at the receptor adenylate cyclase level.
In contrast to these acute antiphosphaturic effects of vitamin D metabolites, we have observed that vitamin D administration to hyperparathyroid rats with chronic renal failure increases phosphaturia despite blunting urinary cAMP excretion (22). Furthermore, acute 25(OH)D3 administration to parathyroidectomized (PTX) rats receiving infusion of exogenous dibutyryl cAMP causes increased phosphaturia (17). These findings suggest that 25(OH)D3 actually increases the renal tubular cell sensitivity to both endogenous and exogenous cAMP.
We have therefore reexamined the renal response to chronic vitamin D administration under controlled conditions, and, having ascertained that phosphaturia is indeed increased by continuous vitamin D administration despite blunting of urinary cAMP excretion and decreased filtered phosphate load, we have examined whether this may be due to changes in renal cortex expression of the type II sodium-phosphate cotransporter (NaPi-2) protein and mRNA.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Metabolic studies.
Male Sabra rats, weighing 200 g, were acclimatized for 5 days in
individual metabolic cages with free access to tap water and standard
chow containing 0.69% phosphorus and 0.97% calcium. After baseline
24-h urine collection and blood sampling from tail veins, all animals
underwent acute parathyroidectomy by electrocautery under light ether
anesthesia. Unprimed Alzet osmotic minipumps were implanted
subcutaneously (model 2001, Alzet, Palo Alto, CA). They delivered
bovine 1---34 PTH (Sigma, St. Louis, MO) in 2% cysteine HCl at a rate
of 0.24 U (35 ng/h) and/or 1,25(OH)2D3
(Hoffmann La Roche, Basel, Switzerland) in propylene glycol at a rate
of 2.5 ng (6.25 pmol) · 100 g body
wt1 · 24 h
1.
Groups studied. Experiments involved the following groups: group I [vehicle+/vehicle (n = 5)]; group II [vehicle+1,25(OH)2D3 (n = 6)]; group III [PTH+vehicle (n = 6)]; and group IV [PTH+1,25(OH)2D3 (n = 6)].
The animals were returned to their metabolic cages for a further 7 days. Daily body weight, water and food intake, and urine volume were noted. Urine for cAMP excretion measurements was collected under ice-cooled conditions. Urine and blood samples were analyzed spectrophotometrically for creatinine, calcium, and phosphate by using a computer-directed analysis system (Cobas-Mira Roche, Basel, Switzerland). Urinary cAMP was measured in duplicate by the protein-binding assay of Gilman (6) using a RIA [3H]cAMP kit (Amersham, Buckinghamshire, UK).Northern blots. On the death of the animals 7 days after PTX, total RNA was immediately extracted from renal cortex of three rats from each group of rats using a Tri-reagent kit (Molecular Research Center, Cincinnati, OH). The mean biochemical values of these rats were similar to those of the group from which they were selected. Aliquots of 10-20 µg total RNA were resolved electrophoretically on 1% agarose gels under denaturating conditions (formamide/formaldehyde). Nucleic acids were transferred to nylon membranes (Gene Screen; New England Nuclear Research Products, Boston, MA) and cross-linked by ultraviolet irradiation. Membrane strips were hybridized for 16-20 h with 32P-labeled rat-specific PTH/PTH-related peptide (PTHrP) receptor cDNA under stringent conditions. Membranes were washed and autoradiographed by standard procedures. Bound cDNA probes were removed by 15 min of immersion in boiled 1× standard sodium citrate+0.1% SDS, and the same membranes were hybridized with 32P-labeled NaPi-2 cDNA and washed and autoradiographed, the probes were removed again, and membranes were rehybridized with a control probe of 18S ribosomal cDNA.
The radioactive probes were prepared with a Rediprime DNA labeling kit (Amersham) using an EcoR1 fragment of rat PTH/PTHrP receptor cDNA, a full-length cDNA probe of NaPi-2, and a cloned fragment of 18S ribosomal RNA as templates. Binding was quantified by phosphorimaging (Fujis, BAS 1000) and expressed as the ratio of intensities obtained by hybridizing the same strip with PTH/PTHrP receptor and 18S or NaPi-2 and 18S.Preparation of brush-border membranes. Contralateral kidneys from the three rats selected for the Northern blots were rapidly removed from the rats, and slices were cut at 4°C from the superficial cortex, homogenized in a buffer consisting of 300 mM DL-mannitol, 5 mM EGTA, 16 mM HEPES, and Tris, pH 7.5, containing protease inhibitor cocktail tablets (Boehringer Mannheim). Brush-border membranes (BBM) were precipitated from this homogenate by Mg2+ precipitation and differential centrifugation as described before (13). The final pellet was resuspended in the same buffer as above. Protein concentration of the BBM preparation was determined by an automated pyrogallol red calorimetric method (Cobas-Mira Roche), and equal amounts of protein (60 µg) were added to each lane of the polyacrylamide gels.
SDS-PAGE and immunoblotting.
Aliquots of BBM were denaturated 1:1 with sample buffer containing 4%
SDS, 20% glycerol, 1% -mercaptoethanol, and 125 mM Tris · HCl, pH 6.8. Sixty micrograms of BBM protein per lane
were separated on 10% polyacrylamide gels and electrotransferred onto nitrocellulose paper. Protein loading equality between the lanes was
confirmed before chemiluminescence examination by staining with Ponceau
S stain. After blockage with 5% fat-free milk powder, Western blotting
was performed with antiserum against the COOH-terminal amino acid
sequence of NaPi-2 at a dilution of 1:5,000 (10, 14).
Blotting was also performed by using antibodies to the sodium-glucose
cotransporter (SGLT-1; from Alpha Diagnostics, San Antonio, TX) and the
sodium-sulfate cotransporter (NaSi-1) (16). The secondary
antibody was goat anti-rabbit IgG at a dilution of 1:10,000. Antibody
binding was visualized by using enhanced chemiluminescence, and
densitometry was done by phosphorimaging.
Statistics. Results are presented as means ± SE. Analysis of variance was performed for statistical evaluation among the four groups. Results between individual groups were compared by a nonpaired Student's t-test with a modified level of significance according to the Bonferroni method (7).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Metabolic data.
Table 1 shows baseline data after animals
were acclimatized in metabolic cages before PTH minipump insertion. The
groups were similar with respect to body weight, plasma creatinine,
phosphate, and calcium, and creatinine clearance and fractional
excretion of phosphorus (FEphos).
|
|
|
|
|
NaPi-2 protein and mRNA levels.
Figure 3 shows the results of renal
cortex NaPi-2 mRNA levels obtained at day 7 in the four
groups of animals studied. Administration of
1,25(OH)2D3 or PTH both caused decreased
expression of NaPi-2 mRNA. The coadministration of both continuous
1,25(OH)2D3 and PTH infusions significantly
decreased expression of NaPi-2 mRNA more than either agent when infused
alone. Housekeeping gene expression (18S mRNA) was unaffected by any
treatment. The expression of PTH receptor mRNA (Table 3) was
significantly reduced by continuous infusion of PTH (group
III). However, this reduction was not evident after
coadministration of continuous 1,25(OH2)D3.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have previously found that vitamin D metabolites have an acute antiphosphaturic effect on hormone-induced phosphaturia (2, 4, 18). This effect appears to be mediated by interference with the activation of the adenylate cyclase-cAMP receptor complex. Thus it is of great interest that in a more chronic setting, 1,25(OH)2D3 appears to increase the PTH-induced phosphaturia despite a decrease in urinary cAMP.
The obvious explanation might have been simply that in a chronic setting, vitamin D increased intestinal calcium and phosphate absorption and that the increased phosphaturia is a direct consequence of increased phosphate load. However, it may be noted that when we administered 1,25(OH)2D3 alone at a dose that has been reported to increase to normal the flow of intestinal calcium and phosphate absorption in thyroparathyroidectomized rats (20, 21), neither urinary excretion nor fractional excretion of phosphate changed significantly.
Thus explanations other than intestinal phosphate load to explain increased phosphaturia were sought. 1,25(OH)2D3 reduced urinary cAMP in PTH-infused animals after 7-day infusion, thus suggesting that a lessening of the vitamin D blunting effect on PTH-induced adenylate cyclase activity was not the explanation for the increased phosphaturia at 7 days. Notwithstanding, the possibility that increased intestinal uptake of phosphate could be involved at least partly in phosphaturia cannot be ruled out.
Of interest is our finding that the PTH receptor mRNA was downregulated from the PTX state by continuous infusion of PTH and that this was abolished by coinfusion with 1,25(OH)2D3. Others have also found that 1,25(OH)2D3 upregulates the PTH receptor in renal distal tubular cells (23). This might possibly lead to increased sensitivity to the infused PTH. However, urinary cAMP excretion was decreased by coadministration of 1,25(OH)2D3, thus ruling out increased sensitivity to PTH and activation of the adenylate cyclase-protein kinase A pathway. We cannot rule out that the lack of blunting of PTH receptor mRNA in 1,25(OH)2D3+PTH-treated animals may have maintained phosphaturia at high levels via non-adenylate cyclase-dependent mechanisms such as protein kinase C activation.
As noted previously, we also were careful not to induce hypercalcemia in the experimental animals because this is another factor that is known to influence renal phosphate handling (1, 19).
An alternative explanation of the phosphaturic effect of 1,25(OH)2D3 in PTH-infused PTX animals might be via a change in the activity or amount of the major sodium-phosphorus transporter in the rat kidney cortex, i.e., NaPi-2. Our findings suggest that chronic continuous infusion of both 1,25(OH)2D3 and PTH decreases the NaPi-2 mRNA expression in the renal cortex of PTX rats and that their coadministration significantly decreased the NaPi-2 mRNA expression still further. Western blotting of BBM preparations from the renal cortex of the same rats showed that NaPi-2 protein levels paralleled the NaPi-2 mRNA expression. Continuous chronic infusion of PTH or 1,25(OH)2D3 reduced NaPi-2 protein content in the cortex of PTX rats, and their coadministration reduced NaPi-2 protein content still further.
Others have found that renal NaPi-2 protein are increased two- to threefold in chronic PTX rats (10). Acute PTH infusion rapidly reduced BBM NaPi-2 protein. There was little or no effect on NaPi-2 mRNA expression, and it was determined that the rapid decrease in BBM NaPi-2 protein abundance was mediated by acute internalization (endocytosis) of NaPi-2 protein (25). In contrast to the determination of the relatively acute effects of PTH administration, the effect of continuous chronic PTH infusion on renal NaPi-2 mRNA and NaPi-2 protein content have not been previously determined.
We have found an additive effect of chronic 1,25(OH)2D3 infusion to further decrease renal cortex NaPi-2 expression and protein content. This has not previously been reported, and its mechanism is unknown. It appears to be specific for phosphate transport because both NaSi-1 and SGLT-1 proteins were unaltered by the PTH and 1,25(OH)2D3 infusions.
Vitamin D stimulates intestinal Na-Pi cotransporter expression (11), whereas in some cells such as osteoblasts phosphate transport may be inhibited by 1,25(OH)2D3 (8). In vitamin D-deficient rats 1,25(OH)2D3 has been reported to increase juxtamedullary cortex expression of NaPi-2 mRNA and protein, but NaPi-2 expression was decreased in the superficial cortex (24). Our finding that continuous 1,25(OH)2D3 infusion given for 7 days decreases NaPi-2 mRNA and protein expression in PTX PTH-infused rats may suggest a direct effect of vitamin D in decreasing renal NaPi-2 such as has been shown for chronic glucocorticoid administration (9,14, 15), or perhaps there is secretion of an unknown phosphaturic hormone secondary to increased intestinal absorption of phosphate.
It is of mechanistic interest that chronic infusion of 1,25(OH)2D3 alone decreased NaPi-2 expression (Figs. 3 and 4) but that we were unable to demonstrate phosphaturia in these animals (Fig. 2B, Table 2). This is in contrast to both groups of PTH-infused animals in whom phosphaturia increased. Thus it would appear that the presence of PTH is permissive to the chronic physiological effects of vitamin D, a finding that we have previously described for the acute antiphosphaturic effects of vitamin D (4, 17, 18). Others have noted that changes in Na-Pi cotransporter expression are not always correlated well with changes in phosphate transport (12). PTH action may therefore also involve changes in Na-Pi cotransporter activity or in the activity or availability of other transporters that may influence phosphate transport.
The schema in Figure 6 shows the
disparate effects of acute and chronic administration of 25(OH)vitamin
D3 derivatives on PTH-induced phosphaturia. The mechanism
of acute antiphosphaturia appears to be via blunting of adenylate
cyclase activation. Despite blunting of adenylate cyclase activation,
chronic administration of vitamin D derivatives increases phosphaturia
and appears to reduce renal cortex NaPi-2 mRNA and NaPi-2 protein
levels. The cellular mechanisms and the signaling processes by which
this occurs remains to be determined.
|
![]() |
ACKNOWLEDGEMENTS |
---|
The antibody to NaSi-1 was kindly provided by Dr. H. Murer.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: M. M. Friedlaender, Nephrology and Hypertension Services, Hadassah University Hospital, PO Box 12000, Jerusalem, Israel 91120 (E-mail: fried{at}cc.huji.ac.il).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 27 July 2000; accepted in final form 16 May 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bonjour, JP,
and
Fleisch H.
Calcium supply and renal handling of phosphate.
Min Elect Metab
3:
261-267,
1980[ISI].
2.
Brezis, M,
Wald H,
Shilo R,
and
Popovtzer MM.
Blockade of renal tubular effects of vitamin D by cycloheximide in the rat.
Pflügers Arch
398:
247-252,
1983[ISI][Medline].
3.
Friedlaender, MM,
Darmon D,
Wald H,
and
Popovtzer MM.
The in vivo and in vitro effect of calmodulin antagonists on the renal actions of 25(OH)vitamin D3 in the rat.
Pflügers Arch
415:
372-380,
1989[ISI][Medline].
4.
Friedlaender, MM,
Kornberg Z,
Wald H,
and
Popovtzer MM.
Renal effect of vitamin D metabolites: evidence for the essential role of 25(OH) group.
Am J Physiol Renal Fluid Electrolyte Physiol
244:
F674-F678,
1983[ISI][Medline].
5.
Friedlaender, MM,
Wald H,
Kopolovic J,
and
Popovtzer MM.
Colchicine does not interfere with the antiphosphaturic effect of 25(OH)vitamin D3 in the rat.
Miner Electrolyte Metab
14:
103-109,
1988[ISI][Medline].
6.
Gilman, AG.
A protein-binding assay for adenosine 3',5'-cyclic monophosphate.
Proc Natl Acad Sci USA
67:
305-312,
1970[Abstract].
7.
Godfrey, K.
Comparing the means of several groups.
New Engl J Med
313:
1450-1456,
1985[Abstract].
8.
Green, J,
Luong KV,
Kleeman CR,
Ye LH,
and
Chaimovitz C.
1,25-dihydroxyvitamin D3 inhibits Na(+)-dependent phosphate transport in osteoblastic cells.
Am J Physiol Cell Physiol
264:
C287-C295,
1993
9.
Guner, YS,
Kiela PR,
Xu H,
Collins JF,
and
Ghishan FK.
Differential regulation of renal sodium-phosphate transporter by glucocorticoids during rat ontogeny.
Am J Physiol Cell Physiol
277:
C884-C890,
1999
10.
Kempson, SA,
Lötscher M,
Kaissling B,
Biber J,
Murer H,
and
Levi M.
Parathroid hormone action on phosphate transporter mRNA and protein in rat proximal tubules.
Am J Physiol Renal Fluid Electrolyte Physiol
268:
F784-F791,
1995
11.
Kitai, K,
Miyamoto K,
Kishida S,
Segawa H,
Nii T,
Tanaka H,
Tani Y,
Arai H,
Tatsumi S,
Morita K,
Taketani Y,
and
Takeda E.
Regulation of intestinal Na+-dependent phosphate co-transporters by a low-phosphate diet and 1,25-dihydroxyvitamin D3.
Biochem J
3:
705-712,
1999.
12.
Lederer, ED,
Sohi SS,
and
Mcleish KR.
Parathyroid hormone stimulates extracellular-regulated kinase (ERK) activity through two independent signal transduction pathways: role of ERK in sodium-phosphate cotransport.
J Am Soc Nephrol
11:
222-231,
2000
13.
Levi, M,
Baird BM,
and
Wilson PV.
Cholesterol modulates rat renal brush border membrane phosphate transport.
J Clin Invest
85:
231-237,
1990[ISI][Medline].
14.
Levi, M,
Shayman JA,
Abe A,
Gross SK,
McCluer RH,
Biber J,
Murer H,
Lötscher M,
and
Cronin RE.
Dexamethasone modulates rat renal brush border membrane phosphate transporter mRNA and protein abundance and glycolipid composition.
J Clin Invest
96:
207-216,
1995[ISI][Medline].
15.
Loffing, J,
Lötscher M,
Kaissling B,
Biber J,
Murer H,
Seikaly M,
Alpern RJ,
Levi M,
Baum M,
and
Moe OW.
Renal Na/H exchanger NHE-3 and Na-PO4 cotransporter NaPi-2 protein expression in glucocorticoid excess and deficient states.
J Am Soc Nephrol
19:
1560-1567,
1998.
16.
Lötscher, M,
Custer M,
Quabius ES,
Kaissling B,
Murer H,
and
Biber J.
Immunolocalisation of Na/SO4-cotransport (NaSi-1) in rat kidney.
Pflügers Arch
432:
373-378,
1996[ISI][Medline].
17.
Popovtzer, MM,
Mehandru S,
Saghafi D,
and
Blum MS.
Interaction between PTH, vitamin D metabolites and other factors in tubular reabsorption of phosphate.
Adv Exp Med Biol
103:
11-19,
1978[Medline].
18.
Popovtzer, MM,
Robinette JB,
DeLuca HF,
and
Holick MF.
The acute effect of 25-hydroxycalciferol on renal handling of phosphorus: evidence for a parathyroid hormone dependent mechanism.
J Clin Invest
53:
913-921,
1974[ISI][Medline].
19.
Popovtzer, MM,
Robinette JB,
McDonald KM,
and
Kuruvila CK.
Effect of Ca2+ on renal handling of PO
20.
Rizzoli, R,
Fleisch H,
and
Bonjour JP.
Role of 1,25 dihydroxyvitamin D3 on intestinal phosphate absorption in rats with normal vitamin D supply.
J Clin Invest
60:
639-647,
1977[ISI][Medline].
21.
Rizzoli, R,
Fleisch H,
and
Bonjour JP.
Effect of thyroparathyroidectomy on calcium metabolism in rats: role of 1,25-dihydroxyvitamin D3.
Am J Physiol Endocrinol Metab Gastrointest Physiol
233:
E160-E164,
1977
22.
Rubinger, D,
Wald H,
and
Popovtzer MM.
25-Hydroxycholecalciferol and 1,25-dihydroxycholecalciferol enhance phosphaturia in rats with reduced renal mass: evidence for a PTH-dependent mechanism.
Miner Electr Metab
16:
348-354,
1990[ISI].
23.
Sneddon, WB,
Barry ELR,
Coutermarsh BA,
Gesek FA,
Liu F,
and
Friedman PA.
Regulation of renal parathyroid hormone receptor expression by 1,25-dihydroxyvitamin D3 and retinoic acid.
Cell Physiol Biochem
8:
261-277,
1998[ISI][Medline].
24.
Taketani, Y,
Segawa H,
Chikamori M,
Morita K,
Tanaka K,
Kido S,
Yamamoto H,
Iemori Y,
Tatsumi S,
Tsugawa N,
Okano T,
Kobayashi T,
Miyamoto K,
and
Takeda E.
Regulation of type II renal Na+-dependent inorganic phosphate transporters by 1,25-dihydroxyvitamin D3-. Identification of a vitamin D-responsive element in the human NaPi-3 gene.
J Biol Chem
273:
14575-14581,
1998
25.
Traebert, M,
Roth J,
Biber J,
Murer H,
and
Kaissling B.
Internalization of proximal tubular type II Na-Pi cotransporter by PTH: immunogold electron microscopy.
Am J Physiol Renal Physiol
278:
F148-F154,
2000