Marshall L. Stoller, Maxwell V. Meng-Urinary Stone Disease

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80 Mandel and Mandel

two or more components. The admixture of calcium oxalate or apatite accounts for 45%

(15) to 9.2% (5,17) and the incidence of pure whewellite stones varies from 24.9% (11)

to 4% (25). Although the actual percentages vary among the different studies, the most

frequent stone compositions are whewellite/weddellite, pure whewellite, apatite/

whewellite/weddellite, and apatite/struvite. Calcium oxalate was present in 73 ± 7% of

all stones analyzed in these studies.

Because most stones are multicomponent, the method employed in the analysis of

stone material should be capable of resolving all components of the stone, especially all

the crystalline components. The literature on stone analysis methods clearly supports the

use of XRD or FTIR as the prime choices. The use of both methods to obtain supportive

and the most accurate compositional data is ideal. For XRD and FTIR, the accuracy of

the analysis is very strongly dependent on the quality of standard spectra. Most labora-

tories conducting stone analyses prepare their own standards libraries. Unfortunately,

many analysis laboratories use patient stone material to create their standard spectra. As

their stones are analyzed by the same method as they are using to analyze other stone

samples, their unknowns become their standard. As virtually no stone is composed of

only one pure crystalline component, such spectral libraries are very inaccurate and the

potential for skewed and inaccurate stone analysis is highly probable. Preparation of

synthetic stone components for the generation of standards and verification of compo-

sition by alternative methods is the only correct way to prepare a standards library for

either XRD or FTIR, especially for FTIR. Commercial libraries should only be used for

supplemental data in those rare instances when experimental data cannot be correlated

with defined stone component standards, especially for identification of nonbiologic or

false stones.

One issue not yet resolved in the literature is the level of accuracy one should accept

in analysis reports. Specifically, rank order of compositional analysis in multicompo-

nent stones analyzed by XRD is probably the best one can expect. In FTIR analyses,

although it is possible to generate computer aided admixed spectral standards at any

percent admixture, many years of experience and cross-correlating analysis results with

accurately weighed admixtures of pure standards suggests that percent compositional

analysis should be quoted at 10% or more for accuracy.

The issue of stone composition analysis accuracy is certainly clouded by the frag-

mented samples now seen by most analysis labs with the common utilization of litho-

tripsy. Virtually no laboratories can be assured that they have received the entire voided

sample or if the sample they have received for analysis accurately represents overall

stone composition. Accuracy of analysis by other methods such as microscopic evalu-

ation that is quoted as approaching or exceeding the XRD or FTIR levels should be

questioned for scientific basis.

In conclusion, high resolution XRD and FTIR spectroscopic methods of analysis are

currently the only two methods that should be considered for the accurate and complete

characterization of crystals in urine and for the compositional analysis of kidney stones.

Frequently, the use of both XRD and FTIR is beneficial and occasionally necessary for

an accurate analysis of multicomponent stones. The accuracy of these methods of analy-

sis is very much dependent on the quality and completeness of a standards library as well

as on the experience of the instrument operator, especially in the use of computer-based

match routines supporting both XRD and FTIR instruments.

Chapter 5 / Kidney Stones 81

ACKNOWLEDGMENTS

This work was supported in part by the Department of Veterans Affairs as Merit

Review grants (NM), a VA Research Career Scientist Award (NM), local and national

funding for the National VA Crystal Identification Center, and by National Institutes of

Health grants DK30579 (NM), DK62739 (NM), and DK64616 (NM).

REFERENCES

1. Mandel N. Urinary tract calculi. Lab Med 1986; 17: 449–458.

2. Mandel NS, Mandel GS. Physicochemistry of urinary stone formation. In: Renal Stone Disease,

(Pak CYC, ed.). Martinus Nijhoff, Boston, MA, 1987; pp. 1–24.

3. Herring LC. Observations on the analysis of ten thousand urinary calculi. J Urol 1962; 88: 545–

562.

4. Sutor DJ, Scheidt S. Identification standards for human urinary calculus components, using crys-

tallographic methods. Br J Urol 1968; 40: 22–28.

5. Mandel NS, Mandel GS. Urinary tract stone disease in the United States veteran population. II.

Geographic analsysis of variations in composition. J Urol 1989; 142: 1516–1521.

6. Hesse A, Molt K. Technik der infrarotspektroskopischen harnsteinanalyse. J Clin Chem Clin Bio-

chem 1982; 20: 861–873.

7. Berthelot M, Cornu G, Daudon M, Helbert M, Laurence C. Computer-aided infrared analysis of

urinary calculi. Clin Chem 1987; 33: 2070–2073.

8. Lehmann CA, McClure GL, Smolens I. Identification of renal calculi by computerized infrared

spectroscopy. Clinica Chimica Acta 1988; 173: 107–116.

9. Mandel NS, Mandel GS. Structures of crystals that provoke inflammation. In: Advances in Inflam-

mation Research, (Weissmann G, ed.). Raven Press, New York, NY, 1982; pp. 73–94.

10. JCPDS Powder Diffraction Files. International Centre for Diffraction Data. Swarthmore, PA,

1985.

11. Brien G, Schubert G, Bick C. 10,000 analyses of urinary calculi using x-ray diffraction and

polarizing microscopy. Eur Urol 1982; 8: 251–256.

12. Prien EL. Crystallographic analysis of urinary calculi: a 23-year survey study. J Urol 1963; 80:

917–924.

13. Prien EL. Studies in urolithiasis: II. Relationships between pathogenesis, structure, and compo-

sition and calculi. J Urol 1949; 61: 821–836.

14. Lonsdale K, Sutor DJ, Wooley S. Composition of urinary calculi by x-ray diffraction. Collected data

from various localities. I. Norwich (England) and district, 1773–1961. Br J Urol 1968; 40: 33–36.

15. Sutor DJ, Wooley SE. Composition of urinary calculi by x-ray diffraction: Collected data from

various localities. IX. Glasgow, Scotland. Br J Urol 1971; 43: 268–272.

16. Bastian HP, Gebhardt M. Die harnsteinanalyse. Therapiewoche 1976; 26: 5935–5938.

17. Mandel NS, Mandel GS. Urinary tract stone disease in the United States veteran population. II.

Geographic frequency of occurrence. J Urol 1989; 142: 1513–1515.

Chapter 6 / Calcium Physiology 83

METABOLISM

Chapter 6 / Calcium Physiology 85

From: Current Clinical Urology, Urinary Stone Disease:

A Practical Guide to Medical and Surgical Management

Edited by: M. L. Stoller and M. V. Meng © Humana Press Inc., Totowa, NJ

Calcium Physiology

G. Bennett Stackhouse, MD and Marshall L. Stoller, MD

CONTENTS

INTRODUCTION

CALCIUM ABSORPTION FROM THE GASTROINTESTINAL TRACT

BONY STORAGE OF CALCIUM

INTRACELLULAR CALCIUM

EXTRACELLULAR CALCIUM

CALCIUM EXCRETION

HORMONAL FACTORS

CALCIUM RECEPTOR

CONCLUSION

REFERENCES

Key Words: Nephrolithiasis; calcium metabolism; absorption; renal

handling.

INTRODUCTION

An understanding of extracellular calcium handling in the body is helpful for those

who treat stone disease. Calcium is a component in the majority of urinary stones.

Researchers and clinicians seek to broaden their knowledge of calcium handling and of

derangements in calcium metabolism in an effort to uncover the etiology of a patient’s

stone disease and to prevent further recurrence.

Calcium is a divalent cation found in many food sources and is essential for life.

Calcium is important as a building block in bony skeletal crystals, and plays an important

role in multiple physiologic cascades. Extracellular calcium is important to neuromus-

cular excitability, and extreme variations in extracellular calcium concentrations can

lead to tetany (if calcium levels fall) or flaccidity and arrhythmias (if calcium is too high).

Muscular contractions via actin and myosin require intracellular calcium. Calcium also

plays a role in the clotting cascade, especially in converting fibrinogen and prothrombin

to fibrin and thrombin. Many other systems require calcium for normal function. Our

86 Stackhouse and Stoller

understanding of calcium metabolism has grown from clinical studies of pathophysi-

ologic states such as rickets and hyperparathyroidism, from elegant animal physiology

models, and more recently from modern microbiologic techniques. It is important to note

that much of this work has been done with nonhuman tissue sources, but appears to be

applicable to humans. This chapter is a brief summary of current concepts and theory

regarding calcium handling. Further research will be needed to clarify areas that remain

controversial or unclear at present.

Calcium Metabolism Overview

Calcium is absorbed through the intestine wall and stored as apatite crystals in bone

matrix and in intracellular organelles. Calcium is continually liberated from and stored

in bony apatite crystals through osteoblast and osteoclast activity. Homeostasis of cal-

cium levels in both the intracellular and extracellular compartments is maintained within

a very narrow range. Intracellular calcium is generally maintained at 50–100 nM, and

extracellular concentration is 1.1–1.3 mM (8.6–10.5 mg/dL.) Outside this physiologic

range of extracellular calcium concentrations, deleterious effects occur within multiple

systems, most notably the neuromuscular and cardiac systems. Calcium is filtered by the

renal glomerulus and largely reabsorbed in the renal tubule and collecting duct. Calcium

is not secreted by the kidney. Variations in renal reabsorption rates from the urinary

lumen allow for net excretion of calcium from the body. Feedback mechanisms to the

parathyroid gland and to the renal proximal tubule allow control of parathyroid hormone

secretion and vitamin D activation to modify bone turnover, calcium absorption, and net

calcium excretion to maintain homeostasis.

Calcium transport across epithelial surfaces in both the intestine and kidney is a

function of several analogous processes, with similar proteins involved. Both intestinal

calcium absorption and renal calcium reabsorption use active cellular and passive

paracellular systems to move calcium from the lumen (intestinal lumen or renal tubular

Abbreviations

-ATPase Calcium adenosine triphosphatase

CaBP Calcium binding protein, calbindin

CaM Calmodulin

CaR Calcium receptor

CTR Calcitonin receptor

DRI Dietary reference intake

OPG Osteoprotegrin

PMCA Plasma membrane calcium adenosine triphosphatase

PTH Parathyroid hormone

PTHrP Parathyroid hormone-related protein

RANK Receptor activator of NF-κB

RANKL Receptor activator of NF-κB ligand

TRPC Transient receptor potential channels

VDR Vitamin D receptor

VDR

nuc

Vitamin D nuclear receptor

VDR

mem

Vitamin D membrane receptor

Chapter 6 / Calcium Physiology 87

lumen) to the extracellular space. In both enterocytes and renal cells, active cellular

calcium (re)absorption can be divided into three distinct regions: (1) calcium transport

into the cell at the apical (luminal) membrane, (2) transcellular calcium transport to the

basolateral membrane, and (3) calcium extrusion from the cell into the extracellular

compartment. Current research is still defining the similarities and differences of cal-

cium transport across the epithelia of the kidney and intestine (1), as will be shown

subsequently in this chapter.

Calcium Pathophysiology Overview

Derangements of calcium and calcium handling can lead to multiple disease pro-

cesses. Changes in extracellular calcium levels have profound effects. Elevated extra-

cellular calcium can lead to flaccidity, atony, and death. Depressed extracellular calcium

leads to tetany. Defects in calcium handling can lead to dystrophic calcifications, hypo-

or hypercalcemia. Abnormalities of hormonal control of calcium lead to chronic disease

processes. Vitamin D deficiency can lead to rickets and osteomalacia. Hyperparathy-

roidism results in osteopenia, nephrolithiasis, and pathologic fractures. Calcium is also

a prime constituent in the majority of urinary stones in the Western world. Elevated

levels of urine calcium are found in a significant number of patients suffering from stone

disease. Understanding calcium metabolism may be important for prevention and treat-

ment of urolithiasis.

CALCIUM ABSORPTION FROM THE INTESTINAL TRACT

Overview of Gastrointestinal Calcium Absorption

The Western daily diet contains about 1000 mg of calcium. The dietary reference

intake for calcium is 1000 mg/d for adults, 1300 mg/d for adolescents, and 1200 mg/d

for elderly persons (2). About 70% of this daily intake is complexed to sulfates, oxalates,

phosphates, and other moieties in a non-ionic form in the intestinal lumen and cannot

be absorbed. This non-ionized calcium is excreted in the stool. Calcium that remains

ionized or bound to small organic molecules is available for absorption across the

intestinal mucosa through both active and passive processes. This absorption is medi-

ated via cellular and paracellular pathways, as reviewed by Bronner and summarized

here (3). The paracellular pathways involve passive diffusion of calcium down a con-

centration gradient from the bowel lumen into the paracellular spaces. This mechanism

is the predominating means of calcium absorption from the gastrointestinal tract when

total oral calcium intake is high. When total calcium intake is lower, more active absorp-

tion may be required to maintain homeostasis, and the proportional amount of calcium

absorbed through active cellular pathways will increase. Calcitriol serves to regulate

active absorption (see “Transcellular Absorption of Calcium” following) and increases

active absorption in times of low total calcium intake.

Active processes predominate in the duodenum and jejunum, whereas passive absorp-

tion takes place mostly in the ileum but occurs to a lesser extent throughout the intestinal

tract. The proportion of calcium absorbed through passive processes in a given bowel

segment is inversely related to the rate of intestinal propulsion and directly related to the

sojourn time of chyme in that bowel segment. The pH of chyme within differing bowel

segments may also affect how much calcium remains ionized and available for absorp-

tion.

88 Stackhouse and Stoller

Calcium absorption is under hormonal control, with the primary hormone being 1,25

dihydroxy vitamin D (calcitriol). In times of high calcium intake, passive absorption is

favored by the high concentration gradient, and the relatively high serum calcium avail-

ability downregulates calcitriol production. This decreases active absorptive processes.

Paracellular Absorption of Calcium

Paracellular pathways involve calcium movement through tight junctions into narrow

spaces between cells. This is the predominant mechanism of intestinal calcium absorp-

tion, especially when calcium is plentiful in the diet. Calcium ions and small molecules

complexed to calcium are diffusible between cells (4). Tight junctions are found where

epithelial cell borders come into close contact at the apex of the cells, and serve to restrict

paracellular movement and to define apical and lateral surfaces of the cells. They are

made of integral membrane proteins and junctional adhesion molecules. Recent research

suggests that particular ion channels are inserted into tight junctions to facilitate selec-

tive passage of certain ions (5).

Regulation of paracellular movement is not well elucidated. Passive paracellular

movement is influenced by the electrochemical gradient from the lumen to the intercel-

lular space. Factors that indirectly affect passive movement include structural changes

to the tight junctions. Tight junctions are modulated by hormones, growth factors, cyto-

kines, toxins, and possibly phosphorylation (3).

Transcellular Absorption of Calcium

Calcium is absorbed in the intestine via cellular and paracellular pathways.

Paracellular absorption occurs throughout the small and large intestine, but mainly in

the ileum. Cellular absorption occurs mostly in the duodenum and jejunum (6). Cellular

pathways involve three distinct steps for moving calcium ions from the intestinal lumen

across the enterocyte to the extracellular space: (1) calcium transport from the intes-

tinal lumen into the cell, (2) movement across the intracellular space, and (3) extrusion

of calcium out of the basolateral membrane of the cell into the intercellular space

(Fig. 1). This overall process is under the hormonal control of calcitriol, via its inter-

action with the vitamin D receptor (VDR).

RANSPORT FROM THE INTESTINAL LUMEN INTO THE CELL

Because the intracellular calcium concentration is usually lower than the intestinal

lumen calcium concentration, this first step of calcium movement into the cell is most

often down a chemical gradient. This movement must be regulated, as the intracellular

calcium concentrations are very low (50–100 nM) and unregulated movement of cal-

cium from the lumen would rapidly raise the intracellular levels to supraphysiologic

levels. The membrane transport mechanism was originally labeled CaT1. CaT1 appears

to be regulated by calcitriol. Experiments in VDR knockout mice show that calcitriol

dependent absorption accounts for approx 90% of total movement across the cell apical

lumen (7). Further work has identified CaT1 as a transient receptor potential channel,

TRPV6 (8). Other names for TRVP6 in the literature include ECaC2 and CaT-like. A

closely related protein to TRPV6 is TRPV5, which appears to be more important in the

renal epithelium and will be discussed below. TRPV6 is part of a family of transient

receptor potential channels (TRPC). TRPV6 appears to be inactivated by high levels of

intracellular calcium, and at high intracellular calcium concentrations it is bound by

Chapter 6 / Calcium Physiology 89

calmodulin (CaM). The activity of TRPV6 appears to be modulated by S100A10-

Annexin2 complex. S100A10 complexes with Annexin2 as a heterotetramer, and is

present in multiple cellular processes. It appears to have a regulatory function for TRPV5

and TRPV6 (9). Binding and regulation of these TRPCs may help control calcium entry

into the cell.

Kinetic studies by Bronner et al. show that movement of calcium across the apical cell

membrane may be via a dual mechanism—a carrier mediated mechanism that involves

use of active transport molecules to move calcium across the cell membrane, and a

channel mediated mechanism that relies on opening and closing porous channels to

allow calcium to diffuse into the cell along electrochemical gradients (3). Subsequent

work implies that the dynamics attributed to CaT1 correspond well to the carrier medi-

ated portion of the dual mechanism postulate of calcium absorption. The channel medi-

ated portion is regulated by intracellular calcium concentration. The channel mediated

mechanism has not been verified, and further work is needed to determine the signifi-

cance of this “dual mechanism” in humans.

ALCIUM MOVEMENT ACROSS THE CELL TO THE BASOLATERAL MEMBRANE

After entry into the cell, calcium is transported across the cell to the basolateral

membrane. This must be tightly regulated to allow extrusion from the basolateral mem-

brane at the same rate as influx from the apical membrane, to avoid changes in intra-

cellular calcium level. Evidence indicates that >90% of calcium crossing the cytosol is

bound to members of the calcium binding protein family (CaBP), calbindins, during

intracellular movement. Binding to calbindins serves to buffer intracellular calcium

levels, as the bound calcium is not ionic, free, metabolically active intracellular calcium.

Fig. 1. Calcium transport across epithelial cells. Two TRPCs (transient receptor potential chan-

nels) are important for calcium homeostasis. TRPV5 is the dominant form in the renal tubule,

whereas TRPV6 is dominant in intestinal segments. CaBP, Calcium binding protein (calbindin)

refers to a family of calcium binding transport proteins. Calbindin-D9k is predominant in

enterocytes. Calbindin-D28k appears to be more important in renal tubular cells. PMCA1b, a

calcium adensosine triphosphatase. NCX1, sodium calcium exchange protein.

90 Stackhouse and Stoller

The maximum capacity for transcellular calcium transport is directly related to the

calbindin content of the cell. This transport in the intestinal luminal cell involves calcium

binding to calbindin-D 9K. The expression of calbindin-D 9k has been well studied, and

expression in the duodenum is higher than in other intestinal segments. Expression of

calbindin-D 9k is regulated by calcitriol, and decreases in older animals (10). It is not

clear whether calbindin-D 9k binding alone is capable of transporting/buffering calcium

across the cytosol. It appears that calcium may also be transported across the cytosol in

calcium enriched vesicles, formed near the apical membrane via disruption of actin

filaments. Calcium ions then are bound to CaM, and the actin filaments are repaired. The

calcium enriched vesicles fuse with lysosomes and may then be transported across the

cells via a microtubular network.

ALCIUM EXTRUSION INTO THE EXTRACELLULAR SPACE

On reaching the basolateral membrane, the calcium must be extruded from the cell

into the extracellular space. This must occur against an electrochemical gradient. Two

cellular mechanisms have been proposed for this activity. One is a plasma membrane

calcium adenosine triphosphatase pump (Ca

ATPase, specifically PMCA1b), and the

other is a sodium–calcium exchange pump (NCX). In the intestine the majority of cal-

cium is extruded by the PMCA1b. This pump is activated by calcium, CaM, and

calbindin-D. A smaller proportion is extruded by the NCX, a Na

exchange pump.

Three NCX genes have been identified in mammals thus far—NCX1, NCX2, and NCX3.

NCX1 appears to be the active pump in intestinal and renal tissues. In renal tissue it

represents the predominate means of extrusion from the basolateral membrane.

NCX1 is regulated by membrane potential, phosphokinase C activation, hydrogen

ions, nucleotides, and parathyroid hormone (PTH). Direct actions of PTH on the intes-

tine are debated. It is not clear whether PTH augments the NCX1 protein activity or

whether it increases NCX1 mRNA expression. NCX1 mRNA expression is also regu-

lated by calcitriol (11).

Potassium dependent sodium–calcium exchange pumps (NCKX) have also been iden-

tified and are expressed in renal and intestinal tissue, but it is not currently known what

role they play in calcium extrusion at the basolateral membrane (12,13).

Regulation of Intestinal Calcium Absorption

Calcitriol regulates expression of the calcium binding proteins (calbindins) and there-

fore regulates the rate of calcium transport across the cell. Calcitriol also serves to

activate the Ca

-ATPase pump, regulating the extrusion of calcium from the enterocyte

into the extracellular space (14). Classic teaching is that PTH participates only by stimu-

lating calcitriol production, thereby leading to increased intestinal absorption of calcium

and phosphate from the intestine. This has been challenged by some authors who propose

that PTH may have direct effects on calcium uptake and transport in the intestine (15).

See section “Calcium Regulating Hormones” for further discussion of hormonal con-

trols of intestinal calcium absorption.

BONY STORAGE OF CALCIUM

Overview of Calcium Metabolism in Bone

In addition to providing a structural frame for the body, bones serve as a reservoir for

calcium. The total body calcium store is about 1.2 kg. Ninety nine percent of this calcium

Chapter 6 / Calcium Physiology 91

is stored as apatite in bone, and 1% is in cells or in plasma. Bony calcium stores are not

static. The actions of parathyroid hormone on the bony reservoir are crucial for the daily

regulation of serum calcium levels. Bones are continually remodeled through osteoblast

and osteoclast activity. During the course of a typical day, 300 mg of calcium is resorbed

from bone and 300 mg is added to bone. This resorption and new bone formation not only

serves to regulate homeostasis in extracellular calcium levels; it is also critical in bony

remodeling.

Osteoblast Activity

Osteoblast activity lays down new apatite crystals, binding and storing calcium and

phosphate. Calcium is an agonist for osteoblastic activity. Elevated extracellular cal-

cium levels may promote bone formation, however reports of the calciumsensing recep-

tor (CaR) in osteoblasts give conflicting evidence for the importance of CaR in regulation

of osteoblastic activity (16,17). Osteoblastic activity may be regulated by calcyclin, a

calcium-binding intracellular protein (18).

Osteoclast Activity

Osteoclasts are large multinucleated cells formed by fusion of osteoclast precursor

cells. The precursor cells differentiate from hematopoietic stem cells. Osteoclast activity

involves resorption of bone and is well summarized by Väänänen (19). Both calcitriol

and PTH are needed to increase osteoclast activity and mobilize calcium from bone by

breakdown of apatite crystals. Interestingly, calcitriol and PTH also increase absorption

of calcium from the intestine and raise serum calcium levels, thus they also cause more

calcium to become available for new bone formation. Calcitonin inhibits osteoclast

activity and serves to lower serum calcium levels by causing a net increased apatite

formation. CaR is also present in osteoclasts and in osteoclast precursors. Stimulation

of CaR by high levels of calcium inhibits osteoclast activity and increases the rate of

apoptosis (20). Stimulation of CaR in precursor cells appears to inhibit formation of

osteoclasts. Conversely, stimulation of VDR by calcitriol in osteoclast precursors pro-

motes osteoclast differentiation and increases osteoclast numbers (21). Other hormones

are known to affect osteoclast differentiation, including estrogen and macrophage colony

stimulating factor.

Hormonal Regulation of Bony Calcium Stores

Hormonal regulation of bony calcium stores provides for rapid correction of extra-

cellular calcium fluctuations and maintenance of homeostasis. This regulation serves

to increase or decrease the relative activities of osteoblasts and osteoclasts to store

calcium as apatite or to liberate more calcium into the extracellular space. The interac-

tion between osteoblasts and osteoclasts is complex and regulated. Osteoblasts express

receptor activator of NF-κB ligand (RANKL) and they also express and extrude

osteoprotegrin (OPG). OPG functions as a “decoy” ligand for RANKL. OPG can bind

and effectively block RANKL, preventing it from binding to receptor activator of

NF-κB (RANK). Osteoclast precursors express RANK as a surface protein. RANKL–

RANK interaction leads osteoclast precursors to differentiate into osteoclasts. This

RANKL–RANK interaction is blocked by excess OPG. Calcitriol stimulates RANKL

expression and represses OPG production. PTH and prostaglandins also stimulate

RANKL expression (22) (Fig. 2).