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. Author manuscript; available in PMC: 2009 Mar 1.
Published in final edited form as: Semin Nephrol. 2008 Mar;28(2):111–119. doi: 10.1016/j.semnephrol.2008.01.004

ROLE OF INTERSTITIAL APATITE PLAQUE IN PATHOGENESIS OF THE COMMON CALCIUM OXALATE STONE

Andrew P Evan 1, James E Lingeman 2, Fredric L Coe 3, Elaine M Worcester 3
PMCID: PMC2329574  NIHMSID: NIHMS45736  PMID: 18359392

Abstract

Using intraoperative papillary biopsy material from kidneys of idiopathic calcium oxalate, intestinal bypass for obesity, brushite, cystine, and distal renal tubular acidosis stone formers during percutaneous nephrolithotomy, we have determined that idiopathic calcium oxalate stone formers appear to be the special case, though the most commonly encountered one, in which stones form external to the kidney and by processes that do not involve the epithelial compartments. It is in this one group of patients that we find not only abundant interstitial plaque, but also strong evidence that the plaque is essential to stone formation. The initial site of plaque formation is always in the papillary tip, and must be in the basement membrane of the thin loop of Henle. With time plaque spreads throughout the papilla tip to the urothelium, which under conditions we do not understand, is denuded and thereby exposes the apatite deposits to the urine. It is on this exposed apatite that a stone forms as an overgrowth, first of amorphous apatite and then layers of calcium oxalate. This process generates an attached stone fixed to the side of a papilla, allowing the ever-changing urine to dictate stone growth and composition.

Keywords: Loops of Henle, papillary biopsies, hyaluronan

Above all, one can say that stone disease is various. Stones can be composed of calcium oxalate (CaOx), calcium phosphate including apatite and brushite, uric acid, cystine, struvite, drugs, and a host of minor and rare crystals [1]. Within many of these stone groups the causes, proven or suspected, run on to lists of diseases, traits, urine chemistry abnormalities, and diet patterns, so stones can best be called the end product of innumerable causes. Here we are interested in the most common type of stone patient, one whose stones are predominantly on average greater than 50% CaOx and in whom one can exclude with confidence all of the systemic diseases known to cause such stones. These patients are often called idiopathic calcium stone formers (ICSF) although most if not all harbor the genetic trait of hypercalciuria, not so much a disease as one tail of the distribution of urine calcium excretions found in humans [1]. It is in this one group of patients that we find not only abundant interstitial plaque, but also strong evidence that the plaque is essential to stone formation.

1. Origin of plaque

The initial site of plaque formation is always in the papillary tip, and must be in the basement membrane of the thin loop of Henle [2] because that is the one site always involved with plaque when any plaque is present, and is the only site involved with plaque in isolation (Figure 1 panel A). Within the basement membrane plaque are individual particles of alternating mineral and organic layers in a tree ring configuration (Figure 1 panel B). Plaque clearly migrates from the basement membrane into the surrounding interstitium (Figure 1 panel C), and when it does so the individual particles can be found associated in an orderly way on type 1 collagen (Figure 1 panel D). Subsequently, particles associated with type 1 collagen fuse into a syncytium in which islands of mineral appear to float in an organic sea (Figure 1 panel E). We believe this final form of plaque is actually composed of fused particles still in association with collagen, and that the plaque organic matrix, mineral, and collagen are tightly associated (Figure 1 panel F). As is evident in all the panels of this figure, the mineral phase of plaque are always overlaid with organic matrix, so that uncoated mineral is never present.

Figure 1.

Figure 1

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Initial sites of interstitial plaque and its progression. The initial site of interstitial deposits (arrows) is the basement membranes of thin loops of Henle at the papilla tip as seen by light (panel A) and TEM (panel B). They appear as multi-layered spheres (insert panel B). With time these deposits appear to migrate into the near interstitial space (panels C & D) where they are associated with type 1 collagen bundles (arrowheads, panel D). These individual deposits coalesce (double arrows, panel E) to form islands of mineral in an organic sea (panel F). Magnification; x1,800 (A); x30,000 (B & C); x35,000 (D); x30,000 (E); x23,000 (F).

2. Composition of Plaque

The mineral phase of plaque is invariably biological apatite as determined by high resolution FTIR and electron diffraction [2]. Within plaque osteopontin is abundant (Figure 2 panel A). Within individual particles, osteopontin (OP) localizes preferentially at the interface between the apatite and the adjacent organic matrix [3] (Figure 2 panel B). The third heavy chain (H3) of the inter-alpha trypsin molecule (ITI) is present in plaque [4] (Figure 2 panel C). Within individual particles, H3 localizes within the organic matrix layers (Figure 2 panel D), a site different from that of OP. H3 is also present in the interstitium, more abundant in ICSF than in normals (Figure 2 panel C) and co-localizes with hyaluronin (HA). OP is well known to slow the growth, aggregation and nucleation of CaOx [5–8]. The ITI complex itself also is known to inhibit crystallization [9–11]. All molecules that affect crystallization tend to adhere to crystals, so the localization of OP at the mineral interface is not surprising. Whether H3 itself affects crystals is not known.

Figure 2.

Figure 2

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Immunohistochemical localization of osteopontin and ITI heavy chain 3 in interstitial plaque. Both osteopontin (arrows, panel A) and heavy chain 3 of ITI (double arrows, panel C) are localized to the islands of interstitial plaque. However, within single deposits, immunoelectron staining of osteopontin found at the interface of the crystalline material and the organic matrix (dark dots, panel B) and heavy chain 3 only in the matrix layer (dark dots, panel D). Magnification; x100 (A); x 30,000 (B); x130 (C); x40,000 (D).

3. Mechanisms fostering plaque

The abundance of plaque can be quantified using intra-operative digital imaging, and plaque expressed as percent coverage of the papillary surfaces [12] (Figure 3). In patients for whom such quantification was performed, 24-hour urine samples collected at times random and remote from the surgery show a strong positive correlation of plaque abundance with urine calcium excretion, and strong negative correlations with urine volume and pH (Figure 4). Although we have no information about ion compositions in the interstitial micro-environment where plaque forms, these data suggest that high interstitial calcium concentrations are created by a combination of hypercalciuria and water conservation, and perhaps interstitial fluid pH is increased by urine acidification leading to formation of apatite in suitable matrix such as the thin limb basement membrane. How calcium might concentrate near the thin limbs is unknown, but the vas recta are very close to the limb so that their basement membranes are nearly in apposition, suggesting the vessels may be very important in the process.

Figure 3.

Figure 3

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Digitized image of papillary plaque. The papillary border was outlined (panel A) in order to measure the total number of pixels encompassed within the papillary domain. Next the individual sites of plaque were outlined (panel B) so that the total pixel number could be measured within the plaque area domain. Reprinted with permission.12

Figure 4.

Figure 4

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Urine correlates of papillary plaque. Fractional plaque coverage per papilium varies inversely with urine volume (upper left panel) among stone formers (closed circles) and non-stone forming control subjects (open circles). Plaque coverage varies with urine calcium excretion (upper middle panel) and is inverse to urine pH (upper right panel). A composite multivariate regression score using urine volume and calcium excretion (lower left panel) and one that includes urine pH as well (lower right panel) strongly correlates with plaque coverage. Reprinted with permission.12

4. Evidence that CaOx stones grow on plaque

Perhaps the most obvious evidence is simple observation; CaOx stones are readily on plaque (Figure 5 panel A) from which they can be removed (Figure 5 panel B) leaving the original growth location bare (Figure 5 panel C). Others [13] have found evidence of plaque on stone surfaces. In a retrospective analysis of stone attachment, about 48% of stones were clearly on plaque at the time of removal; this figure is an underestimate because efforts were not consistently made to document the material stones were attached to [14]. Clinical support comes from the fact that the number of stone formed, adjusted for the duration of stone disease is proportional to the surface coverage by plaque [15], what one would expect if plaque were essentially nucleating stones, or offering a secure lodging place for their growth.

Figure 5.

Figure 5

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Digital image of a papilla from an ICSF patient obtained with an endoscope at the time of percutaneous nephrolithotomy. Panel A shows numerous sites of Randall’s plaque (irregular white areas at arrows) as well as an attached stone (asterick). Panel B shows this stone being removed while panel C shows this same papilla post stone removal. This stone appears to have been attached to sites of Randall’s plaque (double arrows).

5. Mechanism for stones to grow on plaque

En bloc biopsy of very small stones [16] permits us to demonstrate the anatomy and microstructure of the plaque – stone interface (Figure 6 panels A and B). Ultra-structure at the old attachment site (Figure 7 panel A) reveals loss of urothelial cells; above the tissue, in the old urinary space, rafts of crystals (arrows) are imbedded in a homogeneous grey matrix accompanied by cell debris (arrowheads). Higher resolution of the region within the square (Figure 7 panel B) reveals that the exposed plaque is covered by a dark ribbon-like layer of alternating lamina of crystal – white in this figure – and black organic material (arrow). Crystals extend from the rafts into the outer surface of the ribbon (arrowheads). Higher resolution of the square (Figure 7 panel C) shows masses of tiny crystals growing directly in the outer ribbon (asterisk), the attachment site of one of the large raft crystals (arrows), and the crystals within the inner ribbon layers – in white. The blow-up inset shows the micro-crystals within the inner lamina of the ribbon; one can count 4 white and 5 organic layers in this specimen. At the same resolution, at another location, one again sees the large raft crystals imbedded in their homogeneous matrix (double arrows) and masses of crystals growing in the outer layer of the ribbon (arrow).

Figure 6.

Figure 6

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Human Kidney Stone. Two stones adhere to a papillum of a CaOx stone former (panel A, patient 1, digital intra-operative endoscopic image). During PNL the larger stone (arrow) was removed en bloc with its underlying tissue (panel B, light microscopy). The edge of a region of Randall’s plaque is visible just under the stone at the tip of the arrow (panel A). The plaque is seen more clearly in the biopsy specimen (panel C), at the arrow. Reprinted with permission.16

Figure 7.

Figure 7

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TEM Images of the Tissue Attachment Site. Randall’s plaque in tissue at the attachment site (lower portion of panel A, TEM image) presents a sharply demarcated boundary to the original urine space (upper portion of panel A), which contains several rafts of large crystals (arrows) and cell debris (arrowheads). One raft lies closer to the surface (within the square) and a large crystal (within the circle) extends from the raft to the tissue surface. At higher magnification of the square in panel A (panel B) many more crystals of this raft are seen to extend to and reach the plaque surface (within the square of panel B). A particularly large crystal within the circle of panel A, is highlighted here (panel B) by small arrowheads. The plaque boundary has the appearance of a multilayered ribbon (single arrow at left). At higher magnification (panel C) the plaque boundary has nine separate layers (small square of panel C and square insert at upper right), in which five thin black organic lamina alternate with four, white lamina. In the thickest of the white lamina one can see tiny thin spicules that run perpendicular to the surface and have the appearance of multiple voids that contained tightly packed crystals (small arrows, insert, panel C). Large numbers of small crystals are growing into the outer border of the ribbon (asterisk) and merge with more peripheral large crystals that are embedded in a homogeneous gray matrix in what was the urine space. Double arrows highlight a large in-growing crystal. The region marked ‘B’ in panel B reveals the same pattern (panel D) of tiny crystals growing into the plaque border and merging with large crystals embedded in a homogeneous matrix and extending into the urine space. Double arrows mark a large crystal already highlighted by small arrowheads in panel B; its relationship to large numbers of smaller crystals that eventually merge into the plaque border (arrow) is apparent. Very large sharp edged crystals in what was the urine space are surrounded by a homogeneous grey matrix (arrowheads). Magnification; x1,200 (A); x1,800 (B); x8,800 (C); x8,800 (D). Reprinted with permission.16

On the stone itself, one finds large rectangular crystals (arrows) at the interface similar to those in the raft (Figure 8 panel A). As one moves upward, into the bulk of the actual stone and away from the interface, these big crystals give way to masses of small crystals typical of stone architecture, and all imbedded in matrix (asterisk). The large crystals are in a matrix (Figure 8 panel B) that is quite different from plaque in being more homogeneous (arrow) and lacking the large voids that plaque has because of its islands of apatite. At this magnification such islands would be larger than one of the large crystals.

Figure 8.

Figure 8

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TEM Images of the Stone Attachment Site. Large lath shaped crystals are seen embedded in a featureless gray matrix (panel A) that closely resembles the rafts in Figure 7. At higher magnification of the region at the arrow in panel A, the matrix appears coarsely granular (panel B) and does not contain the characteristic round voids of Randall’s plaque. Magnification; x1,200 (A); x1,800 (B). Reprinted with permission.16

This is not a movie, merely a single set of still pictures, but the sequence of events seem rather obvious. Plaque is exposed because urothelial cells either are damaged or undergo apoptosis; this step requires new research. After exposure the plaque is overlaid with new matrix. Tiny crystals form in the new matrix, in successive waves, forming the ribbons. At some point the rate and quantity of crystal formation permits explosive growth outward so instead of lamina one finds heaping up of crystal into an anchored stone. This heaping up would extend the stone from the large initial crystals into the masses of smaller crystals as illustrated in Figure 8.

Evidence supports this sequence [16]. Another en bloc biopsy includes a stone so small it could be sectioned with only limited demineralization. Therefore, staining could identify crystals and permit outlining of the original interface (Figure 9, panel A, dotted white lines). FTIR spectra reveals biological apatite within plaque (Figure 9 panel B) as expected; at the interface itself (Figure 9 panel A, dotted lines) we found not biological apatite but an amorphous apatite (Figure 9 panel B). At Area 1, the closest to the interface (Figure 9, note both panels) we found biological apatite; at Area 2, midway toward the stone periphery, we found a mixture of biological apatite and CaOx. At Area 3, toward the periphery of the stone, we found pure CaOx. These findings are exactly what one would predict from the sequence proposed in the prior paragraph.

Figure 9.

Figure 9

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Light Microscopic Images of the Stone – Tissue Interface and micro-FTIR analysis. In the Yasue stained section (panel A), kidney tissue (bottom of panel A) contains Randall’s plaque accumulations (arrows) near the stone interface cross the interface and merge into the mineral of the attached stone (Area 1). Stone mineral extends continuously from region 1 into the stone interior (Areas 2 and 3). Micro-FTIR analysis of the tissue section in panel B revealed the characteristic band for hydroxyapatite (asterisk, panel B) for the large mass of plaque (arrows, panel A). The interface itself (between white dotted lines, panel A) revealed a broadened band (†, panel B) characteristic of amorphous apatite. Area 1 (panel A) revealed the typical hydroxyapatite band (#, panel B). In Area 2 (panel A) bands of hydroxyapatite and CaOx (arrowhead, tracing marked area 2, panel B) both were detected. Finally, in Area 3, toward the urine space border of the stone panel A, μ-FTIR revealed only CaOx (panel B, arrowhead). Reprinted with permission.16

Immuno-histochemistry reveals that OP is present in the stone and plaque, as expected, and crosses the interface without discontinuity [16]. Tamm – Horsfall protein, known to be restricted to the urine and thick ascending limb of the loop of Henle, is present only on the urine space side of the interface, and extends to the interface surface. From these findings we presume that the organic material forming the ribbon overlay on exposed plaque comes from urine molecules adsorbed initially onto the matrix of plaque. As new crystals nucleate in this urine matrix, the crystals themselves can attract molecules that have affinities for them, thereby creating the new stone.

6. Unique character of ICSF pathology vs. all other forms to date

What we have shown here has been found to date in no other type of stone forming patient but the ICSF. Patients whose stones contain brushite (calcium mono-hydrogen phosphate) have plaque [17], but their inner medullary collecting ducts (IMCD) and ducts of Bellini (BD) are often plugged with apatite crystals; cell death and interstitial fibrosis are usual with deposits. Attached stones on plaque are not found. Patients with intestinal bypass for obesity and CaOx stones have no plaque; they have IMCD apatite plugging, as in brushite stones, though milder [2]. Patients with apatite stones and renal tubular acidosis resemble those with brushite disease except the plugging and interstitial fibrosis are more diffuse [18]. Patients with cystinuria plug their BD with cystine, but also plug IMCD with apatite [19]. In other words, ICSF appear to be the special case, though the most commonly encountered one, in which stones form external to the kidney and by processes that do not involve the epithelial compartments.

7. Implications for research and clinical practice

Perhaps the most obvious remark is that renal pathology of stone formers will vary with even subtle clinical distinctions – brushite in CaOx stones, for example. So pathology without clinical detail is uninterruptible. The mechanisms producing plaque are crucial to understanding ICSF, and are unknown. A true animal model would be invaluable. For clinicians, the ability to quantify plaque radiographically could prove very valuable in patient care, as we do not know what one can do to prevent it, or even cause some regression. Urologists can now visualize the papillae routinely via flexible ureteroscopy using digital optics; when stones are seen on plaque, the patient is almost certainly an ICSF, and when IMCD and BD are seen to be plugged the diagnosis must be otherwise. Conversion from CaOx to apatite or brushite indicates a parallel conversion from plaque and attachment to IMCD and BD plugging with distinctive renal disease, so treatment efforts should be increased. How to avoid such conversion is not known.

Acknowledgments

Supported in part by NIH grant PO1 DK56788

Footnotes

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