bove all, one can say that stone disease isvarious. Stones can be composed of cal-cium oxalate (CaOx), calcium phosphate
ncluding apatite and brushite, uric acid, cys-ine, struvite, drugs, and a host of minor and rarerystals.1 Within many of these stone groups t h eauses, proven or suspected, run on to lists ofiseases, traits, urine chemistry abnormalities,nd diet patterns, so stones can best be calledhe end product of innumerable causes. Heree are interested in the most common type of
tone patient, one whose stones are predomi-
Department of Anatomy and Cell Biology, Indiana University School ofMedicine, Indianapolis, IN.
Methodist Hospital Institute for Kidney Stone Disease, Indianapolis, IN.
Nephrology Section, University of Chicago, Chicago, IL.upported in part by National Institutes of Health grant PO1 DK56788.ddress reprint requests to Andrew P. Evan, PhD, Chancellor’s Professor,Department of Anatomy and Cell Biology, Indiana University School ofMedicine, 635 Barnhill Dr, MS5035, Indianapolis, IN 46202-5120. E-mail:[email protected]
270-9295/08/$ - see front matter
W2008 Elsevier Inc. All rights reserved. doi:10.1016/j.semnephrol.2008.01.004
eminars in Nephrology, Vol 28, No 2, March 2008, pp 111-1
antly on average greater than 50% CaOx and inhom one can exclude with confidence all of
he systemic diseases known to cause suchtones. These patients often are called idiopathicalcium stone formers (ICSFs), although mostf not all harbor the genetic trait of hypercalci-ria, not so much a disease as one tail of theistribution of urine calcium excretions found
n human beings.1 It is in this one group ofatients that we find not only abundant inter-titial plaque, but also strong evidence that thelaque is essential to stone formation.
RIGIN OF PLAQUE
he initial site of plaque formation is always inhe papillary tip, and must be in the basementembrane of the thin loop of Henle2 because
hat is the one site always involved with plaquehen any plaque is present, and it is the only
ividual particles of alternating mineral and or-anic layers in a tree-ring configuration (Fig.B). Plaque clearly migrates from the basementembrane into the surrounding interstitium
igure 1. Initial sites of interstitial plaque and its progres the basement membranes of thin loops of Henle at th
icroscopy. (B) They appear as multilayered spheres (insehe near interstitial space where they are associated witheposits coalesce (double arrows) to form (F) islands o,800�, (B and C) 30,000�, (D) 35,000�, (E) 30,000�
Fig. 1C), and when it does so the individual a
articles can be found associated in an orderlyay on type 1 collagen (Fig. 1D). Subsequently,articles associated with type 1 collagen fuse
nto a syncytium in which islands of mineral
(A and B) The initial site of interstitial deposits (arrows)lla tip as seen by (A) light and (B) transmission electronand D) With time these deposits appear to migrate intoe 1 collagen bundles (arrowheads). (E) These individual
eral in an organic sea. Magnification is as follows: (A)(F) 23,000�.
ssion.e papirt). (C
(D) typ
ppear to float in an organic sea (Fig. 1E). We
bpcm1mon
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Interstitial apatite plaque 113
elieve this final form of plaque actually is com-osed of fused particles still in association withollagen, and that the plaque organic matrix,ineral, and collagen are associated tightly (Fig.
F). As is evident in all panels of Figure 1, theineral phase of plaque always is overlaid with
rganic matrix, so that uncoated mineral isever present.
OMPOSITION OF PLAQUE
he mineral phase of plaque invariably is bio-ogical apatite as determined by high-resolutionourier transform infrared (FTIR) and electroniffraction.2 Within plaque osteopontin (OP) isbundant (Fig. 2A). Within individual particles,P localizes preferentially at the interface be-
igure 2. Immunohistochemical localization of osteopoC) H3 of ITI (double arrows) are localized to the islaB) immunoelectron staining of OP was found at the intots) and (D) H3 only in the matrix layer (dark dots). M
nd (D) 40,000�.
ween the apatite and the adjacent organic ma-rix3 (Fig. 2B). The third heavy chain (H3) of thenter-alpha trypsin molecule (ITI) is present inlaque4 (Fig. 2C). Within individual particles,3 localizes within the organic matrix layers
Fig. 2D), a site different from that of OP. H3lso is present in the interstitium, more abun-ant in ICSFs than in normals (Fig. 2C), andolocalizes with hyaluronin. OP is well knowno slow the growth, aggregation, and nucleationf CaOx.5-8 The ITI complex itself also is knowno inhibit crystallization.9-11 All molecules that af-ect crystallization tend to adhere to crystals, sohe localization of OP at the mineral interfaces not surprising. Whether H3 itself affectsrystals is not known.
nd ITI H3 in interstitial plaque. Both (A) OP (arrows) andf interstitial plaque. However, within single deposits,of the crystalline material and the organic matrix (darkcation is as follows: (A) 100�, (B) 30,000�, (C) 130�,
ntin ands o
erfaceagnifi
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114 A.P. Evan et al
ECHANISMS FOSTERING PLAQUEhe abundance of plaque can be quantifiedsing intraoperative digital imaging, and plaque
s expressed as a percentage of coverage of theapillary surfaces12 (Fig. 3). In patients forhom such quantification was performed, 24-our urine samples collected at times randomnd remote from the surgery showed a strongositive correlation of plaque abundance with
igure 3. Digitized image of papillary plaque. (A) The pixels encompassed within the papillary domain. (B) Nexixel number could be measured within the plaque are
igure 4. Urine correlates of papillary plaque. Fractionolume (upper left panel) among stone formers (�) anaries with urine calcium excretion (upper middle panelultivariate regression score using urine volume and ca
H as well (lower right panel) strongly correlates with plaque
rine calcium excretion, and strong negativeorrelations with urine volume and pH (Fig. 4).lthough we have no information about ionompositions in the interstitial microenviron-ent where plaque forms, these data suggest
hat high interstitial calcium concentrations arereated by a combination of hypercalciuria andater conservation, and perhaps interstitialuid pH is increased by urine acidification, lead-
ry border was outlined to measure the total number ofindividual sites of plaque were outlined so that the totalain. Reprinted with permission from Kuo et al.12
que coverage per papilium varies inversely with urine–stone-forming control subjects (Œ). Plaque coverageis inverse to urine pH (upper right panel). A compositeexcretion (lower left panel) and one that includes urine
apillat the
al plad non) andlcium
coverage. Reprinted with permission from Kuo et al.12
isHlcbv
ES
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Interstitial apatite plaque 115
ng to formation of apatite in suitable matrixuch as the thin limb basement membrane.ow calcium might concentrate near the thin
imbs is unknown, but the vas recta are verylose to the limb so that their basement mem-ranes are nearly in apposition, suggesting theessels may be very important in the process.
VIDENCE THAT CaOxTONES GROW ON PLAQUE
erhaps the most obvious evidence is simplebservation; CaOx stones are readily on plaqueFig. 5A), from which they can be removedFig. 5B), leaving the original growth locationare (Fig. 5C). Others13 have found evidence oflaque on stone surfaces. In a retrospective anal-sis of stone attachment, about 48% of stonesere clearly on plaque at the time of removal; thisgure is an underestimate because efforts wereot made consistently to document the materialo which stones were attached.14 Clinical sup-
igure 5. Digital image of a papilla from an ICSF patieephrolithotomy. (A) Numerous sites of Randall’s plaquetone (asterisk). (B) The stone is removed. (C) The same
ttached to sites of Randall’s plaque (double arrows).
ort comes from the fact that the number oftones formed, adjusted for the duration oftone disease, is proportional to the surfaceoverage by plaque,15 what one would expectf plaque essentially were nucleating stones, orffering a secure lodging place for their growth.
ECHANISM FORTONES TO GROW ON PLAQUE
n bloc biopsy of very small stones16 permits uso show the anatomy and microstructure of thelaque–stone interface (Fig. 6A and B). Ultra-tructure at the old attachment site (Fig. 7A)eveals a loss of urothelial cells; above the tis-ue, in the old urinary space, rafts of crystalsarrows) are imbedded in a homogeneous greyatrix accompanied by cell debris (arrow-eads). Higher resolution of the region withinhe square (Fig. 7B) reveals that the exposedlaque is covered by a dark ribbon-like layer oflternating lamina of crystal (white in Figure
tained with an endoscope at the time of percutaneousular white areas shown by arrows) as well as an attachedla after stone removal. This stone appears to have been
nt ob(irregpapil
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116 A.P. Evan et al
B) and black organic material (arrow). Crystalsxtend from the rafts into the outer surface ofhe ribbon (arrowheads). Higher resolution ofhe square (Fig. 7C) shows masses of tiny crys-als growing directly in the outer ribbon (aster-sk), the attachment site of one of the large raftrystals (arrows), and the crystals within thenner ribbon layers (visible in white). Thelow-up inset shows the microcrystals withinhe inner lamina of the ribbon; one can count 4hite and 5 organic layers in this specimen. At
he same resolution, at another location, onegain sees the large raft crystals imbedded inheir homogeneous matrix (double arrows) andasses of crystals growing in the outer layer of
he ribbon (arrow).On the stone itself one finds large rectangular
rystals (arrows) at the interface similar tohose in the raft (Fig. 8A). As one moves up-ard, into the bulk of the actual stone and away
rom the interface, these big crystals give wayo masses of small crystals typical of stone ar-hitecture, and all imbedded in matrix (aster-sk). The large crystals are in a matrix (Fig. 8B)hat is quite different from plaque, it beingore homogeneous (arrow) and lacking the
arge voids that plaque has because of its islandsf apatite. At this magnification such islandsould be larger than one of the large crystals.This is not a movie, merely a single set of still
ictures, but the sequence of events seemsather obvious. Plaque is exposed becauserothelial cells either are damaged or undergopoptosis; this step requires new research. Af-er exposure the plaque is overlaid with newatrix. Tiny crystals form in the new matrix, in
igure 6. Human kidney stone. (A) Two stones adhentraoperative endoscopic image). (B) During percutaneoloc with its underlying tissue (light microscopy). (A) Thtone at the tip of the arrow. Reprinted with permission
uccessive waves, forming the ribbons. At some t
oint the rate and quantity of crystal formationermits explosive growth outward, so insteadf lamina one finds heaping up of crystal into annchored stone. This heaping up would ex-end the stone from the large initial crystalsnto the masses of smaller crystals as illus-rated in Figure 8.
Evidence supports this sequence.16 Anothern bloc biopsy includes a stone so small it coulde sectioned with only limited demineraliza-ion. Therefore, staining could identify crystalsnd permit outlining of the original interfaceFig. 9A, dotted white lines). FTIR spectra re-eals biological apatite within plaque (Fig. 9B)s expected; at the interface itself (Fig. 9A,otted lines) we found not biological apatiteut an amorphous apatite (Fig. 9B). At area 1,he closest to the interface (Fig. 9, note bothanels) we found biological apatite; at area 2,idway toward the stone periphery, we foundmixture of biological apatite and CaOx. At
rea 3, toward the periphery of the stone, weound pure CaOx. These findings are exactlyhat one would predict from the sequenceroposed in the previously.Immunohistochemistry reveals that OP is
resent in the stone and plaque, as expected, androsses the interface without discontinuity.16
amm–Horsfall protein, known to be restrictedo the urine and thick ascending limb of theoop of Henle, is present only on the urinepace side of the interface, and extends to thenterface surface. From these findings we pre-ume that the organic material forming the rib-on overlay on exposed plaque comes fromrine molecules adsorbed initially onto the ma-
a papillum of a CaOx stone former (patient 1, digitalphrolithotomy the larger stone (arrow) was removed ene of a region of Randall’s plaque is visible just under theEvan et al.16
re tous ne
e edg
rix of plaque. As new crystals nucleate in this
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Interstitial apatite plaque 117
rine matrix, the crystals themselves can attractolecules that have affinities for them, thereby
reating the new stone.
NIQUE CHARACTER OFCSF PATHOLOGY VERSUSLL OTHER FORMS TO DATEhat we have shown here has been found to
igure 7. Transmission electron microscopy images of tttachment site (lower portion, transmission electron mhe original urine space (upper portion), which contarrowheads). One raft lies closer to the surface (within the raft to the tissue surface. (B) At higher magnificationxtend to and reach the plaque surface (within the sighlighted here by small arrowheads. The plaque boundt left). (C) At higher magnification the plaque boundaryight), in which 5 thin black organic lamina alternate witee tiny thin spicules that run perpendicular to the surfaightly packed crystals (small arrows, insert). Large numbibbon (asterisk) and merge with more peripheral largehat was the urine space. Double arrows highlight a largattern of tiny crystals growing into the plaque border aatrix and extending into the urine space. Double arrow
n B; its relationship to large numbers of smaller crystpparent. Very large sharp-edged crystals in what was tharrowheads). Magnification is as follows: (A) 1,200�,ermission from Evan et al.16
ate in no other type of stone-forming patient s
ut the ICSF. Patients whose stones containrushite (calcium monohydrogen phosphate)ave plaque,17 but their inner medullary collect-
ng ducts (IMCDs) and ducts of Bellini (BD)ften are plugged with apatite crystals; celleath and interstitial fibrosis are usual with de-osits. Attached stones on plaque are not
ound. Patients with intestinal bypass for obe-
ue attachment site. (A) Randall’s plaque in tissue at thepy image) presents a sharply demarcated boundary to
everal rafts of large crystals (arrows) and cell debrisare) and a large crystal (within the circle) extends fromsquare in A, many more crystals of this raft are seen to
). A particularly large crystal within the circle of A iss the appearance of a multilayered ribbon (single arrowseparate layers (small square and square insert at upperhite lamina. In the thickest of the white lamina one can
have the appearance of multiple voids that containedf small crystals are growing into the outer border of thes that are embedded in a homogeneous gray matrix inowing crystal. (D) The region marked B reveals the samerging with large crystals embedded in a homogeneousa large crystal already highlighted by small arrowheadst eventually merge into the plaque border (arrow) ise space are surrounded by a homogeneous grey matrix,800�, (C) 8,800�, and (D) 8,800�. Reprinted with
he tissicroscoains she squof thequareary hahas 9h 4 wce anders o
crystale in-grnd mes markals thae urin
ity and CaOx stones have no plaque; they have
Iaabiw
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118 A.P. Evan et al
MCD apatite plugging, as in brushite stones,lthough milder.2 Patients with apatite stonesnd renal tubular acidosis resemble those withrushite disease except that the plugging and
nterstitial fibrosis are more diffuse.18 Patientsith cystinuria plug their BD with cystine, but
igure 8. Transmission electron microscopy images ofeen embedded in a featureless gray matrix that closelyhe region at the arrow in A, the matrix appears coarself Randall’s plaque. Magnification is as follows: (A) 1,200�
igure 9. Light microscopic images of the stone–tissuection, kidney tissue (bottom) contains Randall’s plaqunterface and merge into the mineral of the attached sto
into the stone interior (areas 2 and 3). (B) Micro-FTIRor hydroxyapatite (asterisk) for the large mass of plaqueevealed a broadened band (B, †) characteristic of amorand (B, #). (A) In area 2 bands of hydroxyapatite and CaA) Finally, in area 3, toward the urine space border of th
ith permission from Evan et al.16
lso plug IMCDs with apatite.19 In other words,CSFs appear to be the special case, althoughhe most commonly encountered one, in whichtones form external to the kidney and by pro-esses that do not involve the epithelial com-artments.
one attachment site. (A) Large lath-shaped crystals arebles the rafts in Figure 7. (B) At higher magnification ofular and does not contain the characteristic round voids(B) 1,800�. Reprinted with permission from Evan et al.16
rface and micro-FTIR analysis. (A) In the Yasue-stainedumulations (arrows) near the stone interface cross theea 1). Stone mineral extends continuously from regionis of the tissue section revealed the characteristic bandows). The interface itself (A, between white dotted lines)apatite. (A) Area 1 revealed the typical hydroxyapatitearrowhead, tracing marked area 2) both were detected.e, �-FTIR revealed only CaOx (B, arrowhead). Reprinted
the stresemy gran
e intee accne (aranalys(A, arrphousOx (B,e ston
IR
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Interstitial apatite plaque 119
MPLICATIONS FORESEARCH AND CLINICAL PRACTICE
erhaps the most obvious remark is that renalathology of stone formers will vary with evenubtle clinical distinctions—brushite in CaOxtones, for example. So pathology without clin-cal detail is uninterruptible. The mechanismsroducing plaque are crucial to understanding
CSFs, and are unknown. A true animal modelould be invaluable. For clinicians, the ability
o quantify plaque radiographically could proveery valuable in patient care because we do notnow what one can do to prevent it, or evenause some regression. Urologists now can vi-ualize the papillae routinely via flexible uret-roscopy using digital optics; when stones areeen on plaque, the patient is almost certainlyn ICSF, and when IMCDs and BD are seen toe plugged the diagnosis must be otherwise.onversion from CaOx to apatite or brushite
ndicates a parallel conversion from plaque andttachment to IMCDs and BD plugging withistinctive renal disease, so treatment effortshould be increased. How to avoid such con-ersion is not known.
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J Clin Invest. 2005;115:2598-608.2. Evan AP, Lingeman JE, Coe FL, et al. Randall’s plaque
of patients with nephrolithiasis begins in basementmembranes of thin loops of Henle. J Clin Invest.2003;111:607-16.
3. Evan AP, Coe FL, Rittling SR, et al. Apatite plaqueparticles in inner medulla of kidneys of calcium ox-alate stone formers: osteopontin localization. KidneyInt. 2005;68:145-54.
4. Evan AP, Bledsoe S, Worcester EM, et al. Renal inter-alpha-trypsin inhibitor heavy chain 3 increase in cal-cium oxalate stone-forming patients. Kidney Int.2007;72:1503-11.
5. Asplin JR, Arsenault D, Parks JH, et al. Contribution ofhuman uropontin to inhibition of calcium oxalate
crystallization. Kidney Int. 1998;53:194-9.
6. Vernon HJ, Osborne C, Tzortzaki EG, et al. Aprt/Opndouble knockout mice: osteopontin is a modifier ofkidney stone disease severity. Kidney Int. 2005;68:938-47.
7. Wesson JA, Johnson RJ, Mazzali M, et al. Osteopontinis a critical inhibitor of calcium oxalate crystal forma-tion and retention in renal tubules. J Am Soc Nephrol.2003;14:139-47.
8. Worcester EM, Beshensky AM. Osteopontin inhibitsnucleation of calcium oxalate crystals. Ann N Y AcadSci. 1995;760:375-7.
9. Atmani F, Khan SR. Role of urinary bikunin in theinhibition of calcium oxalate crystallization. J Am SocNephrol. 1999;10 Suppl 14:S385-8.
0. Atmani F, Glenton PA, Khan SR. Role of inter-alpha-inhibitor and its related proteins in experimentallyinduced calcium oxalate urolithiasis. Localization ofproteins and expression of bikunin gene in the ratkidney. Urol Res. 1999;27:63-7.
2. Kuo RL, Lingeman JE, Evan AP, et al. Urine calciumand volume predict coverage of renal papilla by Ran-dall’s plaque. Kidney Int. 2003;64:2150-4.
3. Daudon M, Traxer O, Jungers P, et al. Stone morphol-ogy suggestive of Randall’s plaque. In: Evan AP, Lin-geman JE, Williams JC Jr, editors. Proceedings of theFirst Annual International Urolithiasis Research Sym-posium. AIP Conference Proceedings. Melville, NY:American Institute of Physics; 2007. p. 26-34.
4. Matlaga BR, Miller NL, Terry C, et al. The pathogen-esis of calyceal diverticular calculi. Urol Res. 2007;35:35-40.
5. Kim SC, Matlaga BR, Tinmouth WW, et al. In vitroassessment of a novel dual probe ultrasonic intracor-poreal lithotriptor. J Urol. 2007;177:1363-5.
6. Evan AP, Coe FL, Lingeman JE, et al. Mechanism offormation of human calcium oxalate renal stones onRandall’s plaque. Anat Rec. 2007;290:1315-23.
7. Evan AP, Lingeman JE, Coe FL, et al. Crystal-associatednephropathy in patients with brushite nephrolithia-sis. Kidney Int. 2005;67:576-91.
8. Evan AP, Lingeman J, Coe F, et al. Renal histopathol-ogy of stone-forming patients with distal renal tubularacidosis. Kidney Int. 2007;71:795-801.
9. Evan AP, Coe FL, Lingeman JE, et al. Renal crystaldeposits and histopathology in patients with cystine
