Vitamin D and calcium transport

Kidney International, Vol. 40 (1991), pp. 1177—1189

NEPHROLOGY FORUM

Vitamin D and calcium transport Principal discussant: RMIv KUMAR Mayo Clinic and Foundation, Rochester, Minnesota

contained 100 mg of calcium. Radiographs revealed coarsening of trabeculations and pseudofractures of the pubic rami.

Editors JORDAN J. COHEN JOHN T. HARRINGTON NICOLAOS E. MADIAS

Discussion

DR. RAJIV KUMAR (Chairman, Division of Nephrology, Mayo Clinic, and Ruth and Vernon Taylor Professor, Professor

Managing Editor

of Medicine, Biochemistry, and Molecular Biology, Mayo Medical School, Rochester, Minnesota): The patient is a 58-year-old

CHERYL J. ZUSMAN

woman with proximal limb muscle weakness and intermittent

diarrhea for 4 years, hypocalcemia, borderline hypophosphatemia, an increased immunoreactive PTH concentration,

State University of New York at Stony Brook and Tufts University School of Medicine

undetectable vitamin D metabolites, a non-anion gap metabolic

Case presentation A 58-year-old female was referred with complaints of progressive weakness of the lower extremities for more than 4 years, and weakness of the upper extremities, especially on abduction, over 6 months. She

had been evaluated at a local hospital, but no specific neurologic condition was identified; she was referred for further study. The patient had a history of pulmonary tuberculosis that had been treated with a satisfactory regimen. The possibility of recurrence of tuberculosis previously had been considered, and she had been treated with rifampin, 300 mg, twice daily, and ethambutol, 400 mg, twice daily. The physical examination was unremarkable. A history of intermittent

diarrhea and weight loss of 60 pounds over 4 years was elicited. On admission, the laboratory values were: hemoglobin, 10.0 g/dl, with normocytic, normochromic red cell indices and red cell anisocytosis; sedimentation rate, 57 mm in the first hour; serum iron, 41 zg/dl;

total iron binding capacity, 223 g/dl; serum calcium, 8.4 mg/dl; phosphorus, 3.1 mg/dl; serum albumin, 3.7 g/dl; alkaline phosphatase, 1383 lU/liter, with isoenzymes revealing predominantly bone fraction; immunoreactive parathyroid hormone (PTH) (using a carboxyterminal assay), 85 sL equivalents/mi (elevated), 25-hydroxyvitamin D concentration, undetectable (normal, 10—40 nglml); 1,25-dihydroxyvitamin D <5 pg/mI (normal, 20—60 pg/mi); and serum carotene, 30 g/di (normal, 48—200 g/dl). Blood urea was 15 mg/dl (normal 13—45 mg/dl) and serum

creatinine was 0.6 mg/dl (normal 0.6—0.9 mg/dl). The serum sodium concentration was 144 mEq/liter; potassium, 4.2 mEq/liter; chloride, 116 mEq/liter; and bicarbonate, 20 mEq/liter. Arterial blood gases (on room air) were: pH, 7.29; Pa02, 80 mm Hg; PaCO2, 37 mm Hg; and calculated bicarbonate, 18 mEq/liter. Urinalysis disclosed: pH, 7.0;

acidosis with perhaps inadequate respiratory adaptation, and radiologic evidence of osteomalacia. At the time of her initial visit, she was being treated with rifampin. For the purposes of this discussion, let us make the reasonable assumption that the patient has osteomalacia. How did she develop this condition, and what do we know about the physiology of calcium absorption and excretion that might shed some light on the development of osteomalacia in this patient? I will first review normal calcium handing by the normal human and then will discuss the formation of vitamin D, new information on the role of vitamin D in the induction of calcium-binding proteins, and the interac-

tion of such calcium-binding proteins with the plasma membrane calcium pump. In my discussion, I will point out how abnormalities in the integrated calcium-vitamin D systems might have accounted for the disorder seen in this patient. Normal calcium metabolism Under normal circumstances, an adult human ingests approximately 1000 mg of elemental calcium per day; 800—900 mg of

calcium appear in the feces [1]. Of the ingested calcium, approximately 400—500 mg are absorbed, but approximately 300

mg are secreted via intestinal secretions, pancreatic secretions, and bile into the intestinal tract. Thus, net absorption of calcium into the extracellular fluid is approximately 100—200 mg per day. Extracellular fluid calcium equilibrates between the extracellu-

lar fluid pool and the intracellular and bone pools; the latter includes a rapidly exchangeable bone pool and a more stable

bone mineral pool. Plasma calcium is filtered at the glomerulus and, following reabsorption of a considerable amount, approximately 100—200 mg (or the amount reflecting net absorption Presentation of the Forum is made possible by grants from Pfizer, from the intestinal tract) appear in the urine daily in the patient Incorporated; Sandoz, Incorporated; Marion Merrell Dow Incorpo- in a steady state of calcium balance. In the final analysis, total

trace protein and otherwise unremarkable. A 24-hr urine collection

rated; Merck Sharp & Dohme International; and Amgen Incorporated.

© 1991 by the International Society of Nephrology

body calcium balance depends on the absorption of calcium from the intestinal tract and its excretion by the kidney.

1177

Nephrology Forum: Vitamin D and calcium

1178 Normal plasma calcium

Normal plasma calcium

+

+

Decreased plasma calcium

Increased calcium

Release of increased amounts of PIN

4. in renal fractional excretion of Ca

4. PTh release, f CT release

jjijç of renal 25(OH)D3-la-hyclroxylase

j in renal fractional excretion

of Ca

' motszation of bone/soft

tissue — Ca

4.in renal

25(OH)D3-1a-hydroxylase activity

4. mobihzation of bone/soft

tissue * 4. 1.25(OH)D3SynthesiS

f 1.25(OH)2D3 synthesis

Ca

+ calcium absorption

$

__Normal__ plasma I calcium

4. intestinal calcium

calcium

+

f pasma P1

4.

4. in renal

in plasma P1

I

Stimulation of renal 25(OH)D3-1U- hydroxylase

f plasma Ca

25(OH)D3-la- hydroxylase activity

4. intestinal pitosphate

1

absorption and 4. mobIlization of bone! soft tissue phosphate

1 in renal excretion of

phosphate

activity

Ir tir

fin PTH

Fig. 1. Physiologic defenses in hypo- or hypercalcemic states.

Normal plasma P1

+

1

absorption

$

__Normal___ plasma I

Normal plasma P1

4. plasma Ca

+

Increased intestinal

I

,

__Normal___ * plasma 4

f f

4,in PTH

intestinal phosphate absorption and mobilization of bone!

1 4.in renal phosphate

soft tissue phosphate

excretion

Normal____ __a plasma 4

P1

Vitamin D plays a critical role in the adaptation of the body to

P1

Fig. 2. Physiologic defenses in hypo- or hyperphosphatemic states. CT, calcitonin.

cause, increased amounts of PTH are released into the circula-

decreases in calcium availability. More than 50 years ago, tion [14—24]. Parathyroid hormone increases the mobilization of bone and soft-tissue calcium and also decreases the fractional vitamin in increasing calcium absorption from the intestine excretion of calcium by the kidney. In addition, PTH stimulates [2—7]; even earlier work had shown increased fecal calcium loss the renal enzyme 25-hydroxyvitamin D3-la-hydroxylase, thus in vitamin D-deficient, rachitic children [8—12]. The absorption stimulating the synthesis of 1,25-dihydroxyvitamin D3. This of calcium by the intestinal mucosa requires the expenditure of latter substance increases the intestinal absorption of calcium energy because it occurs against a chemical and electrical and thereby helps bring plasma calcium concentration back gradient [2, 13—24]. The movement of the ion into the cell from toward normal. the intestinal lumen does not require the expenditure of energy Conversely, in hypercalcemia, PTH release decreases, and because it occurs down an electrochemical gradient. At the the release of calcitonin is stimulated. A sequence of events that basolateral portion of the intestinal cell, however, the move- is the converse of that occurring in hypocalcemic states is ment of calcium from within the cell into the extracellular fluid brought into play. It is important to remember that adaptations space occurs against an electrochemical gradient; hence the to alterations in plasma calcium concentrations involve the need for the expenditure of energy. Vitamin D plays a role in secretion of increased or decreased amounts of PTH that, in both of these processes. Excellent evidence demonstrates that turn, alter the renal synthesis of 1,25-dihydroxyvitamin D3. calcium movement into the cell through the apical brush-border Although the synthesis of I ,25-dihydroxyvitamin D3 does inmembrane is facilitated by vitamin D via its active metabolite, crease somewhat in hypocalcemic situations even in the ab1,25-dihydroxyvitamin D3 [2, 13—241. Calcium movement sence of PTH, this change is modest, and it is very likely that across the cytosol of the intestinal cell is enhanced by the the major factor contributing to the altered synthesis of 1,25vitamin D-induced protein, calcium-binding protein [13]; in dihydroxyvitamin D3 in hypo- or hypercalcemia is the change in addition, the vitamin increases the rate of extrusion of Ca2 the plasma concentration of PTH. from within the cell into the extracellular fluid space [13, 16]. The physiologic changes that occur in hyper- or hypophosNicolaysen and others conclusively demonstrated a role for the

The physiologic mechanisms that come into play to maintain normal plasma calcium concentrations involve a complex interaction between parathyroid hormone (PTH) and vitamin D (Fig. 1). If the plasma calcium concentration is diminished due to any

phatemic states are somewhat different than those occurring in states of hypo- or hypercalcemia [14-191 (Fig. 2). If the plasma phosphorus concentration increases, for example, as a result of increased retention of phosphate by the failing kidneys, there is

1179

Nephrology Forum: Vitamin D and calcium

DBP

7-dehydrocholesterol

Pre-vitamin D3

DBP-D3

Vitamin D3

I

Skin

Blood

Fig. 3. The formation of vitamin D3 in the skin. DBP, vitamin-D-binding protein.

OH

Microsomes

— Mitochondria

HO

CH

HO

Vitamin D3

HO

24-

Vitamin D3 25-hydroxylase 25-hydroxyvitamin D3 hydroxylase HO

a reciprocal decrease in plasma calcium concentrations. This

decrease in turn stimulates PTH release, and the renal excretion of phosphate increases. Increased plasma phosphate concentrations also decrease the activity of the 25-hydroxyvitamin D3la-hydroxylase by an unknown mechanism. This enzyme inhibition in turn results in decreased circulating concentrations of 1,25-dihydroxyvitamin D3. Decreased intestinal absorption of

Fig. 4. The formation of / ,25-dihydroxyvitamin D3.

the cholecalciferols becoming de facto vitamins. As is well known, vitamin D1 is formed in the skin from a precursor, 7-dehydrocholesterol, a byproduct in the synthesis of cholesterol. Under the influence of ultraviolet light, the B-ring of 7-dehydrocholesterol is opened to form previtamin D3, which undergoes thermal equilibration to form vitamin D3 [25—38]. Vitamin D3 is transported out of the skin, perhaps in association

phosphate and a decrease in the mobilization of bone and with vitamin-D-binding protein (a member of the a-fetoproteinsoft-tissue phosphate occur as a result. Conversely, when albumin super-family of proteins) to the liver, where it underplasma phosphate concentrations are diminished, renal excre- goes conversion to 25-hydroxyvitamin D3 both in hepatic tion of phosphate decreases as a result of PTH-dependent as microsomes and hepatic mitochondria [38—41] (Fig. 4). The well as PTH-independent mechanisms. Low plasma phosphate microsomal enzyme is operative under normal physiologic concentrations, by a PTH-independent mechanism, stimulate circumstances, whereas the mitochondrial enzyme probably the renal 25-hydroxyvitamin D3-la-hydroxylase enzyme. The resulting increased synthesis of 1 ,25-dihydroxyvitamin D3 in turn increases the absorption of phosphate from the intestine. In addition, more phosphate is mobilized from bone. I would emphasize that adaptations in the vitamin D endocrine system

plays a role when large amounts of vitamin D3 are present in the circulation. The Km for the microsomal enzyme is lower than

that for the mitochondrial enzyme, and the activity of the microsomal enzyme is negatively regulated by the amount of 25-hydroxyvitamin D3 present in plasma. Because the mito-

that occur in response to alterations in plasma phosphate chondrial enzyme is not closely regulated, however, large concentration are independent of PTH.

The formation of vitamin D and its metabolism to active metabolites Vitamin D is not a vitamin in the strict sense of the word inasmuch as it is formed endogenously in sufficient quantities following exposure of an individual to adequate amounts of

amounts of vitamin D3 are converted to 25-hydroxyvitamin D3

in some situations (for example, vitamin D toxicity). The microsomal vitamin D1 25-hydroxylase enzyme is a cytochrome

P-450-like enzyme that consists of a flavo-protein and a cytochrome P-450 that utilizes oxygen to convert vitamin D3 to 25-hydroxyvitamin D3 [20, 42]. The activity of this enzyme is diminished by drugs such as isoniazid [43]. Other cytochrome sunlight [20—221 (Fig. 3). However, inadequate exposure to P-450s that are induced by phenytoin, phenobarbital, and sunlight that has accompanied our way of living has resulted in rifampin decrease concentrations of the substrate, vitamin D3,

1180

Nephrology Forum: Vitamin D and calcium

Table 1. Metabolic clearance rates and production rates of I ,25(OH)2D3 and 24,25(OH)2D3

1 .24,25-trihydroxyvitamin D3 24-oxo-1 ,25-dihydroxyvitamin D3

T112

A-ring monoglucuronide

hours

2326-lactone ?23-oxo-1 ,25-dihydroxyvitamin D3

l,25(OH),D1 24,25(OH)2D3

0.4

44.6 9.2

5.7 1.5

PR pg/day —1.5

26.4

7.2

half-life; MCR, metabolic clearance rate; PR, production rate. Values shown are mean s. a T112,

1,25-dihydroxyvitamin D3

Other polar biliary metabolites

—10

6.5

MCR liter/day

Liver

1 ,25,26-trihy-

droxyvitamin D3 Calcitroic acid 1,25(OH)2D3

Fig. 5. The catabolism of 1,25-dihydroxyvitamin D3.

by accelerating its metabolism to other, inactive compounds [44—49]. The major circulating metabolite of vitamin D3, 25hydroxyvitamin D3 is biologically inert in physiologic amounts. When concentrations of 25-hydroxyvitamin D3 are high, as in hyper-vitaminosis D, 25-hydroxyvitamin D3 can be converted

to other biologically active compounds such as 5,6 trans 25hydroxyvitamin D3 [50]. The intrinsic, albeit low, affinity of 25-hydroxyvitamin D3 for the 1,25-dihydroxyvitamin D3 receptor and the formation of 5,6 trans 25-hydroxyvitamin D1 could

Polar metabolites (e.g., glucuronides)

Reabsorption

Excretion in feces

onjugation Fig. 6. The enterohepatic circulation of 1,25-dihyroxyvitamin LI3.

account for the biologic activity of large, pharmacologic amounts of vitamin D3 or 25-hydroxyvitamin D3 in patients with renal failure and hypoparathyroidism in whom the 25-hydroxy-

strates such as 25-hydroxyvitamin D3 cannot undergo sidechain oxidation in anephric animals [67, 68]. Also, 1,25-dihy-

vitamin D3-la-hydroxylase enzyme activity is low or absent. droxyvitamin D3 undergoes hydroxylation to 1,24,25Under physiologic circumstances, 25-hydroxyvitamin D3 is trihydroxyvitamin D1, a metabolite with reduced bioactivity, in converted in the mitochondria of the renal proximal tubule to target tissues such as the intestine, kidney, and cartilage 1,25-dihydroxyvitamin D3, the biologically active form of the [70—73], In addition, other tissues that contain the 1,25-dihyvitamin [16—18, 20—24, 51—65] (Fig. 4). As might be predicted, droxyvitamin D3 receptor also have 25-hydroxyvitamin D3 or the conversion of 25-hydroxyvitamin D3 to 1 ,25-dihydroxyvita- l,25-dihydroxyvitamin D3 24-hydroxylase activity. Several mm D3 occurs in situations of calcium and phosphate demand. other metabolic processes involving modifications of the side When calcium and phosphate requirements have been met, chain occur in target cells that convert 1,25-dihydroxyvitamin 25-hydroxyvitamin D3 is converted to another metabolite, D3 to other metabolites, such as I ,25,26-trihydroxyvitamin D3, 24,25-dihydroxyvitamin D3, a metabolite of uncertain biologic 23-oxo- I ,25-dihydroxyvitamin D3, 23-oxo- 1 ,25,26-trihydroxyrole [18]. The 25-hydroxyvitamin D3-lct-hydroxylase enzyme is vitamin D3, and a 1,25-dihydroxyvitamin D3-23,26 lactone [16]. a multicomponent cytochrome P-450 complex. It comprises a In general, these processes are induced by I ,25-dihydroxyflavo-protein, an intermediate iron sulfur protein, and a P-450- vitamin D3. They occur in target tissues of the hormone and like enzyme that uses molecular oxygen to convert 25-hydroxy- result in metabolites with reduced biologic activity. They can be vitamin D3 to 1,25-dihydroxyvitamin D3 [60—65]. The activity of thought of as processes that terminate the hormonal response, this enzyme is influenced by a large number of factors. These thereby preventing toxicity. Because modifications of the side have been summarized in two exhaustive reviews [16, 66]. The chain result in metabolites that have reduced biologic activity, most important physiologic regulators are PTH and the concen- it theoretically is possible to design metabolites of I ,25-dihytration of inorganic phosphate in the extracellular fluid. Other droxyvitamin D3 that have increased or prolonged biologic hormones such as calcitonin and growth hormone, and other activity by introducing groups into the side chain that block the ions such as Ca2 ion (direct effect rather than via PTH) and metabolism of the sterol. We recently synthesized an analogue, hydrogen ion may play a role; their role, however, is secondary 26,27-dimethyl-l,25-dihydroxyvitamin D, which has increased to that played by PTH and inorganic phosphorus. and more prolonged biologic activity than 1 ,25-dihydroxyvitaThe metabolism of 1 ,25-dihydroxyvitamin D3 to other sterols mm D3 in vivo [74]. The enhanced bioactivity of this analogue regulates its concentrations in various target tissues. It under- is not due to an increase in its affinity for the I ,25-dihydroxyvigoes side-chain oxidation to form an inert 23-carbon acid, tamin D3 receptor. Several similar analogues have been synthecalcitroic acid [67—691 (Fig. 5). The process is not well regulated sized; one of them, I ,25(OH)2-26--27-hexafluoro-vitamin D3, and accounts for approximately 25% of the metabolism of displays increased biologic activity in vivo and in vitro (see 1,25-dihydroxyvitamin D3. Side-chain oxidation occurs in both references in 74). the liver and intestine and requires a la-hydroxylated substrate, Various side-chain modification processes account for only a fraction of the metabolism of the hormone (approximately 20% namely, 1,25-dihydroxyvitamin D3. Non-la-hydroxylated sub-

1181

Nephro/ogy Forum: Vitamin D and calcium Table 2. Alterations in the enterohepatic physiology of l,25(OH)2D3 in patients with cholestatic liver diseasea

Plasma concentrations

25(OH)D (ng/ml)

I,25(OH)2D (pg/mI)

24,25(OH)2D3

Ca

P04

(ng/mI)

(mg/dl)

(mg/dl)

Normal Primary biliary cirrhosis

17.5 15.7

NSb

NS

0.2 2.6 0.4 0.05

Radioactivity % Total administered

Bile (6 hours)

Urine (6 days)

Feces (6 days)

Normal (N = 5) Primary biliary cirrhosis

15.6 5.7

14

27

69 26

P

1.2

46.1

2.9

42.0

4.5 4.3

1.6

0.3 0.2

9.5

9.4

3.5 3.6

NS

0.5 0.6

NS

iPTH

L

30.9 9 22.6 7.2 <0.01

(N = 2)

a From Ref. 79. b

NS, not significant.

to 30%). In addition other mechanisms, such as biliary excretion of l,25-dihydroxyvitamin D3, also play an important role in the metabolism of the hormone. Following the administration of 1 ,25-dihydroxyvitamin D3, the hormone rapidly clears from the peripheral circulation [75, 76] (Table 1). This phenomenon is associated with the rapid appearance of radiolabel in the bile of animals and humans [77, 781. The metabolites that are excreted in bile normally are reabsorbed in the proximal duodenum and jejunum of animals and human subjects. Thus, there exists an active enterohepatic circulation for 1 ,25-dihydroxyvitamin D3 metabolites (Fig. 6). In certain forms of cholestatic liver disease, such as primary biliary cirrhosis, the biliary excretion of 1 ,25-dihydroxyvitamin D3 is altered and circulating concentrations of PTH are low

Table 3. Distribution of the vitamin D-dependent calcium-binding proteins Type of CaBP

9kD

28kD

Intestine Kidney Placenta Shell gland Pancreas

+ (rodent, bovine, ? human) + (mouse, bovine) + (rat, human)

+ (avian, ? human) + (rodent, human, avian)

+ (porcine)

Brain Bone

— — — —

+ (avian) + (human, avian) + (rodent, human, avian)

—

Teeth Inner ear Visual cortex —

—

+ (avian) + (human, rodent) + (human) + (feline)

(Table 2). The retention of 1,25-dihyroxyvitamin D3 could cause

transiently elevated concentrations of the sterol; these would cause transient hypercalcemia, thus suppressing PTH secre- enterohepatic circulation, as might occur in malabsorption syndromes, could result in disease such as osteomalacia. tion. The lower PTH concentrations would diminish the synthesis of 1,25-dihyroxyvitamin D3. A new steady state (noted in Vitamin D-induced calcium-binding proteins Table 2) would result. These changes could at least partly The manner in which 1,25-dihydroxyvitamin D3 functions in account for the low-turnover bone disease in individuals with various tissues like the intestine or kidney, while not directly primary biliary cirrhosis [79]. relevant to the patient being discussed today, sheds light on the The biliary metabolites of 1,25-dihydroxyvitamin D3 are regulation and mechanism of solute transport in various epithemuch more polar than the native hormone. They are either ha and on the control of growth and cell differentiation. In the neutral or acidic metabolites; the acidic compounds behave like intestine, 1,25-dihydroxyvitamin D3 increases the absorption of glucuronides and sulfates on various chromatographic systems. calcium; it also increases the mobilization of soft tissue and We have isolated in pure form one such biliary metabolite, an bone calcium [15—18, 20—24]. Further, 1,25 dihydroxyvitamin A-ring glucuronide of 1 ,25-dihydroxyvitamin D3 [80]. We have D3 might play a direct role in mineralization, although this is a examined the biologic activities of various model glucuronides matter of considerable debate [18]. Considerable evidence and glucosides and have shown that these compounds are less indicates that 1 ,25-dihydroxyvitamin D3 alters the rate of celactive than the native sterol [79]. They display biologic activity lular growth and differentiation of several cell types; further, it as a result of hydrolysis in the intestine or other tissues of the might have an immune modulatory role [18]. It acts both via glucuronide or glucoside moiety from the aglycone. Further- genomic and non-genomic mechanisms. In target tissues such more, we have shown that they are biologically inactive in as the intestine, it combines with a nuclear receptor that systems that lack endogenous glucuronidase activity. The bil- resembles receptors for thyroid hormone and other steroid iary handling of l,25-dihydroxyvitamin D3 thus can be con- hormones [81—85]. The receptor comprises a hormone-binding ceived of as an excretory route for I ,25-dihydroxyvitamin D3, and a DNA-binding domain; the DNA-binding domain has as the metabolites in bile are less active than the native several "zinc fingers" that may directly associate with seghormone and because a large percentage of them are excreted ments of DNA. Receptor mutations in individuals with vitamin into the feces in an unaltered form. Some of these metabolites D-dependency rickets, type II, have helped define the physiolcan be reutilized after deconjugation in the intestine, as is ogy of the vitamin D receptor and establish its important role in probably the case for the glucuronides. Disturbances in the the mechanism of action of 1 ,25-dihydroxyvitamin D3 [86]. In

1182

Nephrology Forum: Vitamin D and calcium

addition, considerable information suggests that I ,25-dihydroxyvitamin D3 also activates various non-genomic processes such

as inositol phospholipid metabolism. Although there is no

Table 4. Calcium-transporting tissues in which vitamin D-dependent calcium-binding proteins and the plasma membrane calcium pump are present or co-localized

question that these phenomena occur, their precise role in the

mechanism of action of this sterol hormone remains to be Kidney, distal tubule (human, rat) defined.

The hormone receptor domain, probably in association with

other factors, increases the transcriptional activity of RNA polymerase II. Several genes are activated following the administration of 1,25-dihydroxyvitamin D3 [17—19]. The most notable

of the genes with increased activity is the calcium-binding protein (CaBP) gene [19]. These proteins are present in calciumtransporting tissues such as the intestine, the renal distal tubule, and bone, as well as in other non-calcium-transporting tissues

Kidney, other portions Intestine (rat) Placenta (human, rat) Osteoblast-like cells (human) Choroid plexus Shell gland

CaBP

PM Ca2 Pumpa

+

+

—

—

+ + — —

+

+ + + +

a PM, plasma membrane.

such as the pancreas and brain [19, 87—92] (Table 3). Their induction coincides with an increase in calcium transport, and they appear to be induced by vitamin D but not calcium [2, 13], In some tissues (for example, the brain), their amount does not depend on the concentrations of vitamin D. In general, the

region of the protein 1931. The 28 kD CaBP has 6 EF-hand

intestine.

bound to a site that was close to a tryptophan residue. When we

structures, whereas the 9 kD CaBP has 2 EF hands. We studied

the 28 kD CaBP with terbium fluorescence spectroscopy to determine the nature of the interaction between calcium and this protein [941. Fluorescent lanthanides such as terbium CaBPs can be classified into two general groups: a 28 kD CaBP interact with proteins in a manner similar to that exhibited by found in mammalian kidney, brain, and pancreas, and a 9 kD calcium; excitation of terbium bound to the protein by different CaBP found in the placenta and in mouse kidney [191. The latter techniques in the presence or absence of calcium or other protein is also probably found in mammalian intestine. In avian lanthanides allows one to determine the manner in which species, only a 28 kD CaBP exists in several tissues, including calcium binds to the protein and the relationship of different the kidney, brain, intestine, chorioallantoic membrane, and calcium-binding sites to one another. Using lanthanide specbone. In humans, a 28 kD CaBP is present in the distal tubule, troscopy and excitation by direct or indirect methods, we in the brain, particularly in cerebellar cells, and possibly in the showed that the 28 kD CaBP bound between 3 and 4 moles of intestine, whereas the 9 kD CaBP is found in placenta and terbium with high affinity, and that one of the moles of terbium To determine the biologic function of various CaBPs, one performed calcium displacement studies, we observed at least must determine how these proteins interact with calcium, what two classes of binding sites. Using circular dichroism studies, interactions they might have with other enzymes responsible we observed a conformational change in the protein when we for calcium transport, and whether they can increase the added calcium or EDTA. Similarly, intrinsic protein fluoresmovement of calcium across compartments in which they are cence studies demonstrated at least two classes of binding sites present. To do this, one needs to have some idea about the in the protein. Therefore, the model that we propose for the structure of these proteins and their properties following inter- interaction of calcium with the 28 kD CaBP involves a two-step actions with calcium. Many of the studies concerning CaBPs in binding sequence in which either one or two moles of calcium mammalian species—in humans in particular—have been ham- bind to a high-affinity site, and another two moles bind to a pered by the low availability of these proteins and because they relatively lower-affinity site. Similar experiments have been are not easily purified. The chicken 28 kD CaBP and its brain performed by several groups with the 9 kD CaBP demonstrating homologue in mammals, however, are more easily isolated than a conformational change upon binding calcium. are CaBPs from other tissues; these proteins are good models As I noted earlier, the 28 kD CaBPs have 6 EF-hand binding for studying CaBP function, as they are virtually homologous regions, whereas the protein appears to bind only 4 moles of with other human and mammalian 28 kD CaBPs. Likewise, the calcium, We attempted to resolve the apparent dichotomy bovine 9 kD CaBP and the rat 9 kD CaBP from bovine or rat between the numbers of binding sites and the numbers of moles intestine are good models for the human 9 kD CaBPs. To study of calcium bound by the protein. Two of the EF-hand domains the structure of various CaBPs, we isolated the 9 kD CaBP from of CaBP, namely domain 2 and domain 6, are imperfect EF bovine intestine, raised antibodies against it, and used these hands. To study this further, we isolated from rat brain a 28 kD

antibodies to isolate a CaBP clone from a eDNA library

CaBP in native form, and we screened a eDNA library from rat obtained from bovine intestine [91]. We found a high degree of brain for the presence of cDNA clones for the protein [95]. homology among the various mammalian 9 kDa CaBPs [91]. We Analysis of the cDNA clones showed 6 EF hands. Using also isolated chick intestinal CaBP and determined its structure site-directed mutagenesis, domain 2 and domain 6 were re-

using Edman degradation and mass spectrometry [921. We found the amino acid sequence of chick intestinal CaBP to be

moved from appropriate clones. Full-length and mutant proteins then were expressed in large amounts in E. coil. Following

similar to the predicted sequence of chick intestinal CaBP isolation of the proteins, we observed the binding of calcium derived from the nucleotide sequence of a cDNA clone isolated both to the full-length protein and to mutants lacking either one by Hunziker [90]. On examining the structure of these proteins, or both EF hands as noted earlier. Thus, domains 2 and 6 it is clear that they contain EF-hand sequences, described probably are not important for calcium binding. What does all initially by Kretsinger as loop-helix-loop structures that bind this basic information mean clinically, and can we relate it to calcium to various oxygen-containing residues within the loop human disease?

Nephrology Forum: Vitamin D and calcium

CaBPs and the plasma membrane calcium pump Based on its biophysical properties, it is clear that the 28 kD CaBP has biophysical properties similar to those of calmodulin.

1183

is our understanding of the mechanisms involved in the transport of 1,25 dihydroxy vitamin D3 into the nucleus for binding to

its receptor and the subsequent induction of the calciumThe importance of this observation lies in the fact that the binding protein genes? DR. KUMAR: Work is being done on the mechanism of action plasma membrane calcium pump, which extrudes calcium from within the cell, is strongly stimulated by calmodulin. Several of the 1,25 dihydroxyvitamin D receptor. The work falls into 2 groups have tried to determine whether CaBPs increase the general areas: One area of investigation has dealt with abnoractivity of the calcium pump. Unfortunately, the data until now malities that occur in patients who have inherited defects in the have not been clear; in general, most of the studies show that structure of the vitamin D receptor. In essence, mutations CaBP, either bound to calcium or in the absence of calcium, occur either in the 1,25 dihydroxyvitamin D3 binding domain or does not increase or stimulate calcium pump activity [96—103]. in the DNA domain of the receptor; these mutations cause

We have used histochemical techniques to determine

abnormalities in the manner in which the receptor binds to

whether the CaBP protein and calcium pump might be part of a common calcium transporting mechanism. To this end we have co-localized these two antigens in calcium-transporting tissues using immunohistochemistry [104—1101 (Table 4). In human kidney, one finds the calcium pump present only in the baso-

DNA [111—117]. Presumably, a lack of binding to DNA results

in a lack of transcription of CaBP. Less is known about a second area of investigation, namely, how 1,25 dihydroxyvita-

mm D3 binds to its receptor and subsequently increases the transcription of CaBP. Several investigators have examined the

lateral portion of the cells of the distal tubule. The calcium 5' regions of the CaBP gene and have been unable to find pump co-localizes with CaBP in this segment of the nephron. 1 ,25-dihydroxyvitamin D3-receptor specific promoter/enhancer

Similarly, rat distal tubule cells contain both the calcium pump elements in the CaBP gene. Whether or not there are promoter! and CaBP. It is of interest to note that the intercalated cells of enhancer elements in introns of the CaBP gene or in portions the distal tubule in the rat kidney do not contain epitopes for distant from the gene has not been established as yet. Investieither of these proteins. Human osteoblast-like bone cells gators have examined the mechanism of 1,25 dihydroxyvitamin contain calcium pump epitopes and, even though these partic- D3 on the osteocalcin gene [118—120]. As you know, osteocalcin ular cells do not contain CaBP, there is abundant evidence for is a protein formed by osteoblasts; its synthesis is increased by the presence of CaBP in bone. Human placenta contains the 1 ,25-dihydroxyvitamin D3. It appears that the interaction of calcium pump along the basal portion of the syncytiotropho- 1 ,25-dihydroxyvitamin D3 and its receptor occurs at very speblast of the placenta; work performed by other investigators has cific regions (approximately 500—600 base pairs) upstream from shown that the 9 kD CaBP is present in these tissues as well the transcription start site in the osteocalcin gene. Other genes [88]. Similarly, rat duodenum contains substantial amounts of regulated by 1 ,25-dihydroxyvitamin D include the osteopontin CaBP in absorptive cells [89]. It is clear that CaBPs and calcium gene [121]; the same principles that have been observed for the pumps localize together in many tissues. These two proteins are osteocalcin gene apply to the osteopontin gene. very likely part of a common calcium-transporting mechanism. DR. MADIAS: Is there any evidence that the induction of the In the patient we are discussing today, several abnormalities calcium-binding proteins by vitamin D is affected by variables might have contributed to her osteomalacia. The malabsorption such as the age of the animal, and by its calcium, phosphorus, was found to be secondary to bowel bacterial overgrowth. She had malabsorption of dietary vitamin D because of steatorrhea. and vitamin D status? DR. KUMAR: That's an excellent question. There is a resisIn addition, she probably had increased fecal losses of 1,25tance to 1 ,25-dihydroxyvitamin D3 in aging rats that appears to dihydroxyvitamin D3 metabolites as a result of an interruption be due to a decrease in the amount of 1 ,25-dihydroxyvitamin D3 of her enterohepatic circulation. All these problems resulted in receptor [122]. There are also age-related decreases in 1,25low vitamin D, low 25-hydroxyvitamin D, and low 1,25-dihydihydroxyvitamin D3 concentrations that appear to be propordroxyvitamin D concentrations. Additionally, she was taking tional to decreases in GFR that occur with aging [123, 124]. The rifampin, which is known to increase the metabolism of vitamin D3 to other nonactive metabolites, thereby making less sub- decreases in 1,25-dihydroxyvitamin D3 production as well as strate available for the formation of 25-hydroxyvitamin D3. Had receptor concentrations might then cause a decrease in reshe been treated with isoniazid, she also might have had a sponse. DR. JOHN T. HARRINGTON (Chief of Medicine, Newtondecrease in 25-hydroxyvitamin D concentration as a result of suppression of the vitamin D3 25-hydroxylase enzyme. There Wellesley Hospital, Newton, Massachusetts): You mentioned have been descriptions of acidosis affecting 1 ,25-dihydroxyvi- that a reduction in the enterohepatic circulation of bile in tamin D3 concentrations [16], but acidosis is an unlikely culprit patients with primary biliary cirrhosis, and perhaps in this in this particular instance because of its rather modest level. patient with diarrhea, can cause hypocalcemia. Are there any Calcium malabsorption resulted in secondary hyperparathy- diseases in which an associated increase in the enterohepatic roidism. She was treated with small amounts of exogenous circulation in turn might account for some of the hypercalcemic vitamin D3, supplemental calcium, and antibiotics. No evidence states? DR. KUMAR: Not that I'm aware of. of tuberculosis was found. Her bone disease resolved in several DR. PAUL KURTIN (Director, Dialysis Unit, New England weeks. Medical Center): You didn't mention the role of vitamin D in Questions and answers the regulation of PTH. Would you please discuss that? Also, do DR. NIcoLAos E. MAD lAS (Chief, Division of Nephrology, intravenous and oral vitamin D affect PTH regulation differNew England Medical Center, Boston, Massachusetts): What ently?

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Nephrology Forum: Vitamin D and calcium

DR. KUMAR: You're quite right; I neglected to mention that there is good evidence that 1 ,25-dihydroxyvitamin D3 decreases PTH synthesis and that receptors for I ,25-dihydroxyvitamin D3 are present in parathyroid glands [125, 126]; some evidence also suggests that there might be fewer receptors in uremic animals

calcium concentrations that are involved with the function of those cells, That's just speculation.

absorption. Some data show that if you can get the 1,25-

other hydroxylated forms. The other two-thirds would be

dihydroxyvitamin D3 concentrations high enough by oral dosing, you can indeed get suppresssion that might be equivalent to that found with intravenous I ,25-dihydroxyvitamin D3 [129]. I think it's fair to say that the reason intravenous I ,25-dihydroxyvitamin D3 works consistently is that one can get more 1,25dihydroxyvitamin D3 to where it is supposed to get to reproducibly. I don't know of a lot of good data suggesting that the oral form will cause more hypercalcernia because of its direct effect on the mucosa. DR. KURTIN: I have a question regarding some patients we

excreted by the liver, That is the major route of elimination of

DR. HARR!NGTON:

I'd like to return to the quantitative

aspects of vitamin D metabolism. I understand from what you said that side-chain oxidation accounts for 25% of the degradarelative to normal animals [127, 128]. The role of intravenous tion of vitamin D. What accounts for the other 75%? treatment in that regard relates to the fact that one can obtain DR. KUMAR: If you give radioactive 1 ,25-dihydroxyvitamin reproducibly high concentrations of 1 ,25-dihydroxyvitamin D3 D3, eventually all of the tracer will appear in the feces and in plasma and hence in parathyroid cells. This is dificult to do urine. Almost 100% of it enters the feces and urine via excrewhen you give 1 ,25-dihydroxyvitamin D3 orally. That would tory metabolites [133, 1341. I would say that one-third of the suggest that maybe there is a direct effect of I ,25-dihydroxyvi- metabolism of 1,25-dihydroxyvitamin D3 would be accounted tamin D on the intestinal mucosa that increases calcium for by the processes of side-chain oxidation and metabolism to

l,25-dihydroxyvitamin D3. I neglected to mention that the hepatic process is virtually non-saturable. You can give vast amounts of I ,25-dihydroxyvitamin D3 and the same percentage will be excreted no matter what the dose. DR. BESS F. DAWSON-HUGHES (Division of Endocrinology,

New England Medical Center): There is some suggestion that 1 ,25-dihydroxyvitamin D3 affects calcium metabolism through several mechanisms other than the stimulation of active transport of calcium across the intestinal mucosa. One mechanism is see in the dialysis unit and today's patient: is the myopathy that it enhances the passive transport of calcium through the associated with vitamin D deficiency? Do muscle cells have paracellular shunt pathway. Another is that 1,25-dihydroxyvivitamin D receptors, and is the myopathy related to alterations tamin D3 has a trophic effect on the mucosa itself and increases in calcium metabolism or to some other effect? the absorptive surface area. Do you think either of these DR. KUMAR: Vitamin D metabolites have several effects on mechanisms are important in humans? muscle metabolism. A profound hypotonia has been described DR. KUMAR: Let me try to answer the first question on the in individuals who have nutritional vitamin D deficiencies that trophic effects of I ,25-dihydroxyvitamin D3. It's very clear that disappears within a very short period after the administration of I ,25-dihydroxyvitamin D3 has a generalized tonic effect on vitamin D; the restoration of normal muscle tone is not related cells. A vitamin-D deficient animal is smaller than its vitamin to changes in the concentrations of various divalent ions. The D-replete counterpart. If you give vitamin D to a vitamin addition of 25-hydroxy D3 to the medium in which a diaphragm D-deficient animal, the rates of growth dramatically increase is suspended increases the uptake of amino acids and the within 24 hours [135]. Jim McCarthy, a colleague of mine, amounts of amino acid incorporated into muscle protein [130]. examined the morphology of the intestine of vitamin D-deficient These observations could explain alterations in muscle tone in chicks following the administration of 1 ,25-dihydroxyvitamin vitamin D deficiency. Others have demonstrated that 1,25- D3. Rachitic animals had shrunken, atrophic intestinal Inucosa. dihydroxyvitamin D3 alters phospholipid metabolism in muscle The administration of I ,25-dihydroxyvitamin D3 restored the cells [131]. Finally, a group in Oxford has shown that 1,25- mucosa to normal within 12 hours [136]. Also, if you administer dihydroxyvitamin D3 affects the synthesis of troponin C [132]. I ,25-dihydroxyvitamin D3 to a rachitic animal, not only is there Troponin C plays a role in excitation coupling, and it is an E-F an increase in amino acid uptake into calcium-binding protein, hand-like protein in that it binds calcium in a manner similar to there also is an increase in amino acid uptake into all proteins. calmodulin, myosin light chain kinase, and the vitamin D It just so happens that calcium-binding protein synthesis is calcium-binding proteins [132]. Thus, 1,25-dihydroxyvitamin stimulated to a very large extent. D3 has multiple effects on muscle. How these effects relate to DR. DAWSON-HUGHES: Do you think 1,25-dihydroxyvitamin hypotonia is uncertain. Of course, the hypocalcemia found in D3 enhances calcium diffusion/absorption via a paracellular vitamin D deficiency results, on occasion, in seizure disorders. shunt? DR. MADIAS: Let me extend the previous question. You DR. KUMAR: I think that it doesn't. The effect of 1,25mentioned that vitamin D receptors and calcium-binding pro- dihydroxyvitamin D3 is on active calcium transport that reteins exist in many non-calcium-transporting tissues, such as quires energy. I don't believe that paracellular pathways are brain and pancreas. Is there any insight on what their role might energy dependent or are saturable. Clearly, vitamin D-induced be in these tissues? transport is active and saturable, which would suggest that it is DR. KUMAR: I don't know what they are doing in brain. They transcellular as opposed to paracellular. appear to be in very specific parts of the brain, particularly in DR. MADIAS: Is all vitamin D-dependent calcium absorption the Purkinje cells of the cerebellum. I have no good explanation mediated via synthesis of new protein? for what calcium-binding protein is doing in brain. Maybe these DR. KUMAR: To a very large extent, yes. There is some cells require calcium at relatively high concentrations; possibly evidence that vitamin D or 1 ,25-dihydroxyvitamin D3 has calcium-binding protein affects intracellular calcium and free effects on membrane that increase the movement of calcium

Nephrology Forum: Vitamin D and calcium

into the cells. There are non-genomic effects of 1,25 dihydroxy-

1185

DR. HARRINGTON: It is apparent from the data you presented

vitamin D. Norman and coworkers have performed experi- that there is an efficient degradative pathway for vitamin D. ments suggesting that I ,25-dihydroxyvitamin D3 can increase Given those data, how much extra vitamin D could one eat yet the absorption of calcium within 5 minutes in a perfused organ still metabolize it rapidly enough to avoid toxicity? DR. KUMAR: That's difficult to answer in precise quantatitive system [137]. Protein synthesis couldn't play a role that quickly. However, the intestines have to be from vitamin terms. The hepatic excretion of 25-hydroxyvitamin D3 is much D-replete animals. Recent excellent work shows that 1,25- slower than that of I ,25-dihydroxyvitamin D3. It's in the range dihydroxyvitamin D3 increases the uptake of calcium into of 2% to 3% versus 25% to 30%. Clements had a paper in the hepatocytes and osteoblasts [1381. It is uncertain whether Lancet demonstrating that [1401. In terms of how much will transcellular calcium transport is increased in the absence of an have a toxic effect, it depends in large part on stores. If one has increase in protein synthesis. In patients with vitamin D-depen- large stores and takes a modest amount of vitamin D, hyperdency rickets type II, who have a defect in the l,25-dihydroxy- calcemia could result, whereas someone with marginal stores vitamin D3 receptor, severe rickets occurs. Were non-genomic would be able to put a lot of it away and not have problems. I events the only events important in the movement of calcium, can't give you a precise answer, but those are some things you these individuals should not have rickets, but they have very might want to consider. DR. MADIAS: Have all these advances given us any new high circulating concentrations of 1 ,25-dihydroxyvitamin D3. They require 500-600 g of 1,25 dihyroxyvitamin D3 to effect a insights on the mechanism of idiopathic hypercalciuria? Do we cure [1121. There's no question that there are effects of 1,25- know whether hydrochlorothiazide induces calcium-binding dihydroxyvitamin D3 on membrane that are direct and indepen- proteins in the kidney? DR. KUMAR: In renal leak hypercalciuria, the negative caldent of protein synthesis. I remain unconvinced that changes in membrane structure play a major role in the movement of cium balance results in slightly higher 1 ,25-dihydroxyvitamin calcium across the cell; changes in membrane structure may D3 levels. In the patients with absorptive hypercalciuria, there play a role in the uptake of calcium into cells and organelles, but is supposed to be some other factor that increases 1,25whether that is equivalent to absorption of calcium is another dihydroxyvitamin D3 concentrations, perhaps persistent decrements in phosphorus. Does hydrochiorothiazide directly induce story. DR. DAVID H. CAHAN (Division of Nephrology, Faulkner calcium binding protein? I have no knowledge of it doing so. DR. MADIAS: Do we know how vitamin D increases calcium Hospital, Jamaica Plain, Massachusetts): I'm curious about the mechanism of vitamin D toxicity. Given the biologic inac- mobilization from bone? DR. KUMAR: It does so by increasing the activity of osteotivity of 25-hydroxyvitamin D3 and the elegant negative feedback mechanisms on the 1-alpha hydroxylase system of the clasts. Whether 1 ,25-dihydroxyvitamin D3 signals osteoclasts kidney, what is known about 1,25 vitamin D3 levels in vitamin directly or whether it does so via osteoblasts remains to be D toxicity? What is the known mechanism of hypercalcemia? determined. I believe that in physiologic circumstances, bone DR. KUMAR: In vitamin D toxicity, 25-hydroxyvitamin D3 resorption is not stimulated to a significant extent; after 1,25 levels are very high, and 1,25 dihydroxyvitamin D3 concentra- dihydroxyvitamin D3 concentrations enter the higher part of the

tions are either normal or marginally elevated because the normal range, bone resorption begins. I am not aware of hypercalcemia switches off the 1-alpha hydroxylase enzyme. investigators who have been able to segregate cells such as Why then do you have increased calcium absorption? The osteoblasts and osteoclasts and look for receptors for 1,25 25-hydroxyvitamin D3 does bind to the 1,25 dihydroxyvitamin

dihydroxyvitamin D3 in the two different subpopulations as has

D3 receptor, albeit with an affinity that is lower than that of been done for PTH or estrogen. 1,25-dihydroxyvitamin D3. The K0 of 1,25 dihydroxyvitamin D

Reprint requests to Dr. R. Kumar, Nephrology Research Unit, for its receptor is approximately 10 10 or l0 M, whereas the K0 of 25-hydroxyvitamin D3 for the receptor is lO_8 or iO M. Department of Medicine, Biochemistry and Molecular Biology LaboMayo Clinic and Foundation, Rochester, Minnesota 55905, Typically, in vitamin D toxicity, you can have concentrations of ratorv, USA. 25-hydroxyvitamin D3 in the 200—300 ng/ml range, whereas the 1 ,25-dihydroxyvitamin D3 concentration under normal circumReferences stances is only 30—50 pglml. It's feasible that if you have a three-

or fivefold increase in 25-hydroxyvitamin D3 concentrations there might be sufficient 25-hydroxyvitamin D3 to bind with the

receptor to activate events that would cause more calcium absorption. The second explanation is one that we postulated some time ago. We demonstrated that if you administer pharmacologic amounts of 25-hydroxyvitamin D3 to animals, you could detect significant amounts of 5,6-trans 25-hydroxyvitamin D3 in the sera of such animals [139]. Cis-trans isomerization of the triene structure will place the 3-beta hydroxyl group in the position of

the 1-alpha hydroxy group; the increased affinity of the 5,6 trans-25-hydroxyvitamin D3 molecule for the receptor could activate hormonal events that would normally be brought about by 1,25-dihydroxyvitamin D3. Those are the two explanations.

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