This is our main article on Randall’s plaque, a papillary nidus that fosters growth of calcium stones.
Other articles on this site illustrate plaque, and discuss plaque as a mechanism of calcium kidney stone production. But these have used plaque as part of explanations for stones not as something in itself.
This article provides a full narrative about modern plaque research. Because our own research group performed much of the modern work we may seem perhaps partisan. But in fairness we do show what others have contributed.
A word about context.
From long before our time to now, scientists have recognized the massive numerical predominance of idiopathic calcium oxalate stone disease as contrasted with stones arising from systemic disease. Our concern here lies with plaque in such common patients. Elsewhere, and later on, we plan to consider plaque arising from specific diseases. Quite possibly its origins may not be the same in all cases.
In 2014 this review offered a detailed review of publication history concerning plaque. It accords with part of what we have written, partly not. But we are not historians, rather working scientists in the field. And, we did not set out to do a history of it. These authors certainly did not intend a scientific exposition.
The mysterious picture shows Randall’s Plaque embedded in kidney tissue with an attached stone. It is not the first picture of plaque but the most dramatic. I cannot give you access to the article because Journal of Urology is very restrictive.
What Must We Know?
Because our main objective is stone prevention, what must we know about plaque?
The article will tell you it is a common anchoring place for calcium oxalate stone formation, especially in younger patients and perhaps in men more than in women. All studies with more or less enthusiasm identify hypercalciuria as a factor that promotes its development. Our group adds low urine volume and pH. So in the course of stone prevention, control of urine calcium, and maintenance of high urine volume and perhaps pH may well act against new plaque. Urological surgeons can see plaque, and new grading systems permit a kind of rough quantification. Likewise with repeat procedures they may see less plaque in well treated patients.
That is the pragmatic nugget one can extract right now. You may content yourself with it alone, but so barren a statement will mean much more to you once you know the details. Thence this long but pretty text we offer as our contribution.
Who Was Randall?
A Perfected Scientist
Alexander Randall was a professor of urology at University of Pennsylvania.
In 1937, he published this paper that first described the plaque named in his honor.
He deserves this honor for many reasons. Certainly, he founded the theory of kidney stone formation on anchored papillary plaque. Even more, among all who have cultivated the field of stone research, he was, in this work at least, a supreme scientist. He began with a mandarin scholarship, reviewed all contemporary theories of stone production, and found them wanting truth. Perhaps as a result, he experienced the revelation of an inspired idea that he tested well and, thereby, discovered a useful scientific truth.
In other words, he performed the acts of a professional scientist and was rewarded by the joy of discovery. Nothing more can be asked.
His Work Was Long in Coming
Even before this monumental paper, in Feb 1936, we found him lecturing at the N. E. branch of the American Urological Association. Of the discussants Fuller Albright spoke, commenting about his cases of primary hyperparathyroidism with stones. Those stones, he mentioned, were mostly calcium phosphate, and he saw in the urine and kidneys crystal casts of the kidney tubules. To him, the open ends of these casts seemed ideal sites for anchoring.
He was right, Randall was right, but they seemed at odds.
Unabashed, Randall went on to his magisterial discovery, and Albright to his, also. No doubt that audience of distinguished physicians failed utterly to recognize that they had that evening witnessed the passing encounter of the two brightest stars in their universe. And like two stars both passed on their independent ways, drawing around them, each one, his own system of planetary followers. Each founded an entire school of scientific thought.
He Found Prior Theories Wanting
Stasis held that stagnant urine would crystallize. Indeed it will because supersaturated. Yet stones often occurred without stasis, so he rejected the theory as being narrow and mostly inapplicable. Although some believed that Infection might set up a nidus of abnormal tissue that calcified, he noted that sterile stones predominated. Vitamin A deficiency could produce stones in rats, but calcium phosphate, not uric acid or calcium oxalate – the most common one. Even so he gave this theory credence.
Colloidal Chemistry. Here he means excessive amounts of crystal making materials cause stones. In support scientists had given oxamide to rats to raise their urine oxalate, or calcium carbonate to raise calcium. He argues that many people with high urine oxalate form no stones – they knew that then. Likewise in cystinuria stones may not form despite excesses of that amino acid. But ultimately he agrees that supersaturation probably does play a role in determining the type of stone. Parathyroid Hyperfunction we now call primary hyperparathyroidism does cause stones, and he endorsed this as a main factor. He already knew it accounted for only 5% of stones.
He Formed A New Hypothesis and Tested It
Having rejected prevailing theories as either unconvincing or too narrow, he created his own hypothesis about how stones formed.
Hypothesis is a compound of two main items. The first is what I already spoke about – a vision, an imagining about how they might form – in this case on an anchored site not as yet known. His vision was that a site on papilla existed, and stones formed on it. The second part of hypothesis is deduction. If his vision were true, then what necessary prediction might he test in order to falsify it? The test was a search of papilla in cadaver kidneys for a hitherto overlooked site that stones could and did in fact grow on. He looked, he found plaque, and found stones growing on it.
Where Had Everyone Else Been Before Him?
These plaques were not disguised. Any pathologist could have described them. No doubt many before Randall saw them, perhaps made note, but passed by. Surely some observed tiny stones on these plaques. They were not rare nor hard to see. But if you have no idea about what a plaque might mean, how easy to pass it by as trivial, unrelated to what one is looking for. Is that not how things are? We see what we are looking for.
A Word About Science
What Hypotheses Are
I have written about hypothesis in science, and specifically about kidney stones. They consists in useful dreams or imaginings about how nature might have produced what one can plainly observe about the world. All imaginings can satisfy our desire to understand how things got to be the way they are. But useful imaginings do more than satisfy that desire. They make predictions about the world and, therefore, offer us a way to test if the imagining be true. By true I mean predictive of what is there but not known about until imagination pointed the way.
That sequence matters. Only in ignorance would someone imagine the workings of nature in conflict with what is already known. That is why even the most erudite cosmologist cannot imagine usefully about medicine, or botany. Although one always shapes an explanation about how things work around what is already known, most explanations stop short of true prediction about what is not already known but could be looked for.
Randall did not stop short. He imagined how kidney stones might form, looked for precisely what must be found if his imagining were true, and found it there, where he imagined it might be. That is how he discovered plaque as an anchored site for stone formation.
How Hypotheses Create Theory
One hypothesis is not a theory. A theory combines into a coherent story all of the hypotheses that have been proposed, tested, and not falsified. This collection of narrated usable hypotheses is what we ‘know’ about how nature brought about – in this case – kidney stones. For example, Randall sought and tested a new hypothesis to advance the theory of stone production and got us plaque. Later he sought how stones might assemble on plaque. In this his tools limited success. He sought what might create plaque and considered animal experiments that did not yield success. Clearly he hoped for a complete theory that accounted for how plaque formed and how stones grew on it.
He found the plaque, but could not do the rest.
What Exactly Did Randall Do?
A Novel Hypothesis Came to Him
He turned away from contemporary theories with a disdain one can sense 80 years later and focused himself on human tissue in the light of a brilliant hypothesis: “That there must be an initiating lesion that precedes the formation of a renal calculus”. As a further point he identified not the renal pelvis or calyces as host to such a site – too simple and too rugged. Instead, the papillae called to him. Complex, and known for some fragility. From this he made a powerful prediction: “That the initiating lesion was to be looked for on the renal papilla.”
His imagining was of an anchored nidus stones could grow on. His deduction was that such a lesion would be found on the renal papilla. By such a lesion he meant on papilla and with evidence stones grew thereon.
He Tested His Prediction in Human Tissue
If inspiration comes unbidden like a song, the proper test comes from a mastery of method and technique. He inspected autopsy kidneys looking for a site stones had grown on. Finding none would have falsified his hypothesis. As he most brilliantly said, instead of trying to produce a stone, how much better to “…find stone as it occurred in man, and to observe … its point of origin….”
Among 429 pairs of kidneys 73 (17%) showed papillary lesions. These lesions were cream colored, near the tip of the papilla, and definitely inside the urothelial covering. With a microscope it seemed a plaque of calcium in the interstitial tissue, definitely not inside the tubules. He saw that it arose in the basement membranes of the terminal tubule walls and involved the space between the tubules as it grew larger (interstitial means in between the tubules and vessels).
The First Picture of Plaque with Stone
After a number of cases, he found a tiny black stone growing right on the plaque where the lining epithelium had given way so urine could bathe the tissue deposits. Here is his photograph from 1937. The tiny reddish black round dot is in the middle of a white or cream colored clearing on the papilla.
This historic picture should evoke some sense of wonder. The round shape of the image field is because of the old optics in use then for microscopes and magnifying glass. No one before him, before this, had made such an image. So this is discovery incarnadine, the ancient relic of that moment in which dream becomes undeniable reality.
We have both known such moments, rarely enough to remember each one. If life is a warp and weft of common stuff, they shine out like threads of gold. Amidst the endless and so often futile theorizing that litters this field of ours, his image stands forward as the very element of undeniable truth. Of science, the immortal diamond.
First Micrograph of Plaque with Stone Over it
He had a microscope and succeeded in making thin sections of the stone and its underlying plaque. The stain colors calcium, so you can tell where it is.
We are looking at tissue from the place where the stone grew over plaque. The papilla has lost its covering epithelium. The rings of color are around tubules whose lumens remain open. In other words, the calcium deposits in the collagen that supports the tubule lining cells, but does not break through into the tubule to form plugs.
For those not trained in anatomy a special word. Kidney tubules are lined by a single layer of cells. They rest upon a tube of condensed collagen that supports them, their so called basement membrane. Outside that collagen tube run the capillaries that nourish the cells and support their lives. Inside the tubule the cells face into the fluid that the kidneys are converting into final urine.
Plaque forms in those collagen tubes, the basement membranes. Thence, the calcium stain forms rings.
Where the brown stone material lies over plaque, the latter has become very dense. Even so, the open tubules are easy to see.At the end, where the stone layers over it, plaque gets so dense the tubule lumens vanish.
With collaborators, he established the plaque as a salt of calcium crystallized with phosphate and carbonate.
He Uncovered the Plaque Stone Junction
Well enough, you say. I see a stone like material lying over plaque. But, can you show me a real stone with plaque on it where once it attached?
He did that. From chemical analysis the black part proved calcium oxalate, the white part – torn off plaque – proved calcium carbonate and phosphate. This proves that crystals of one kind have grown over crystals of quite another kind. That latter forms in the kidney tissue, in fact in the basement membranes of tubules
He Articulated Modern Understanding of Stone Genesis
“So here we have a definite renal calculus growing on an initiating lesion, and the lesion is a deposition of calcium in the walls of the renal papilla, and the two are of different chemical composition.”
After a brief exposition he goes on to his most remarkable summary:
“This lesion, innocent enough while buried in the wall of a renal papilla, can lose its epithelial covering and from then on be bathed in calyceal urine, and acting as a foreign body, it becomes the nidus upon which urinary salts precipitate. Here, likewise, we can picture the reason how and why renal calculi can remain stationary while increasing in size. Here we can account for a common origin of stone formation, which also allows of the known variation of salts so deposited to form stone, and one can assume that the salt which does crystallize to form a calculus is the one which, at that epoch, is most readily thrown out of suspension.”
Within those quotes lies the articulation of a full theory of kidney stone genesis. At the end, he had proved only a small portion of it but left as his legacy to us the honor of a further attempt.
You would think that after his immense discoveries stone research would follow as schooners in the wake of an ocean liner, or rabbits in the wide path cleared by a passing bear. It was not that way. People remembered his plaque but failed to connect his work directly to their own. I mean by this he discovered because he studied human tissue, discarding as equivocal what could be found in animals or produced in animals by unnatural means. Likewise he discarded elaborate inquiries into chemistry or cells in culture that sought explanations not rooted determinedly in human tissue findings.
As a result we created animal models of stone formation that were utterly correct within their scope but divergent from human tissue for the most part as related to the common calcium oxalate stone. Likewise we successfully uncovered factors that could foster crystallization – supersaturation, regulation of urine calcium, oxalate, citrate and pH, principles of crystallization. But we did not link our discoveries to tissue consequences in the one relevant animal – ourselves.
Work After Randall
Long Before Us
This dramatic picture was taken by W. A. D. Anderson in 1940. Although we have the original paper, neither of us can figure out exactly how he produced this image. It is obviously a histological section but how he solved the problem of cutting mineralized tissue and retaining anatomical contiguity is unclear. Even so this shows without question a stone physically growing on tissue embedded plaque. It is x600 magnification, and so is very tiny. The Von Kossa stain he used stains calcium selectively.
Like Randall, he studied cadaver tissue, and reports that he found plaque in 180 of 1500 autopsies. He could not link plaque to clinical history of kidney stones but noted that records often were sparse.
Vincent Vermooten in the South African Medical Sciences Journal 1940 6:48-49 describes Snow-capped papilla in human kidney. Neither of us can find this reference.
Before us, others tried to identify sites of plaque origin and refine knowledge of its crystal structure. Khan and his colleagues identified plaque from a single patient as hydroxyapatite. A report by Marshall Stoller described plaque as extending far higher up in the medulla than Randall had found, and localized in basement membranes of collecting ducts and vasa recta. In this he agreed with Randall that plaque lay in basement membranes, but unlike Randall also placed it within the long hairpin thin loops of capillaries that descend into the medulla. As a clinical investigator he associated it with hypertension but not with kidney stones.
In a more clinical work, Stoller (I invariably name studies for their major scientist as opposed to casual issues of author sequence) contributed massively as the first person I know of to map plaque on papilla during stone surgery. He found it in a majority of stone formers and specifically showed it as more common among stone formers than people without stones. A second report of quite a few patients (143) linked plaque specially to calcium oxalate stone formers. Among them, those with high urine calcium had more plaque, suggesting an association between them.
What Did We Do Different?
If we contributed at all it was by linking ourselves closely to Randall’s work, and to the groundbreaking observations Stoller made, but with more modern and elaborate instruments and techniques. We did not so much discover anew as refine their work into modernity.
Instead of using autopsy tissues as almost all others had done, we perfected intraoperative biopsy of the papilla to get fresh tissue. Because we did that, our tissues lacked the inevitable degeneration that follows death. Likewise we had immediate clinical information from living patients to correlate with the tissues. We used electron microscopy Randall never knew, and high resolution crystal identification within the tissues themselves. We advanced Stoller’s work through biopsy.
What Did We Want to Know?
Instruments are senseless, mere objects. Science lives in the imagination that glimpses what might be and in the intellect that seeks to prove the worth or not of such a brief vision. We sought not plaque itself, but its source. We reasoned that kidneys produce plaque in the course of their physiological functioning, not as a product of some primary disease, and that the origin point of plaque in the tissue coupled with a deep knowledge of how kidneys worked would lead us to a hypothesis that linked plaque to kidney function.
So, we sought the origin point of plaque in biopsies from stone formers taken in the course of stone removal surgery.
How Could You Know The Origin?
By tracking backward to the tiniest specks, too small for any but an electron microscope to disclose. There, where almost nothing was, must be its origin.
What Did We Find?
We never published this set of
images. The large one on the left of the panel shows a whole kidney with papilla outlined by white circles. We have removed the collecting system and the outer parts of the calyces so the papillae are easy to see.
The leftmost papilla connects to insert ‘B’ through a white line. The arrows in B point to a white material at the papillary tip. This is plaque as Randall would have seen in his autopsy tissues.
The other two papilla have different amounts of plaque. The one labelled A has minimal amounts – at the arrows. That labelled C has more than A and less than B. Moreover in C the plaque lies in four small separate areas.
We noticed this variability of plaque amount throughout our subsequent surgical work, but here it is evident within a complete single kidney.
Studies During Surgery
Like Randall, we found plaque with stones growing on it. Here is a picture taken during surgery. The white plaque stands out. The arrowhead points to an eroded area where a stone may have once lodged.
A low power view of the papillary biopsy shows more or less what he had shown. Our optics are more brilliant, our stain a sharper black.
There are the rings of calcium surrounding open tubules. Some plaque ranges to the very outer rim of the surface. An arrow points to it.
So far, nothing differs from him. We found what he found, exactly. This was a stone former, who made calcium oxalate stones.
The tissues gave no evidence of injury or inflammation. He found the same. It was as if the crystals filled in the collagen that lies between the tubules and blood vessels and specially filled the areas around tubules making rings.
Nearly eighty years later, with better optics and fresher tissues, we replicated his work to a cipher.
Where It Began
Here, in the thin limbs of Henle’s loop. It begins as tiny laminated microspheres of alternating layers of crystal (white) and organic molecules. An arrow in the picture at the right shows such a sphere whose layers stand out clearly. As this is an electron micrograph, the sizes are very tiny indeed.
What are the loops of Henle? For those not anatomists, we have nephron pictures. This is a simple drawing to orient you. Here is a fancier drawing. The loops run down the length of the papilla and help concentrate the urine. Can one imagine a better place to look for calcifications?
With only planar microscopy it is hard to orient plaque in three dimensions within kidney tissue. To do this we used high resolution micro CT imaging of the tiny tissue biopsy material.
In the left panel of the plate below, the double parallel arrows point to a tubule seen along its long axis. A pair of rails lies perpendicular to the arrows, just like tram tracks. These are the outer edges of a tubule covered with a shell of plaque. The paired right hand image shows the plaque in white outline where the tubules pass though as voids. So the plaque collection is around the outsides of the tubules.
The two non parallel arrows at the lower part of the left image point to a tubule with an open lumen at its center and a shell of plaque
outlining its circumference. One cannot see it on the right side panel.
So plaque forms sheaths around tubules which can merge into one another to form the thin shell like structures seen on the right side. Nowhere did we see any mineral plug the tubule lumens. It was all outside.
You might wonder at the lack of a reference to publication of this lovely picture. In fact, one of us (AE) produced it but used it only in a chapter of a book yet to achieve final publication.
What Did We Do Next?
With an origin in hand, we began to test the main points of a complete theory of kidney stone genesis on plaque. What renal functions might produce it, how certain are we that plaque is necessary for stones, how stones form on plaque, all these became our focus.
We Measured Plaque Abundance
Scientists measure things, and we learned to measure the abundance of plaque in humans by making movies of their papilla during kidney stone surgery and calculating the fraction of the papilla covered by plaque by counting pixels inside and outside of plaque borders.
Asked What Urine Measurements Correlated with Plaque
The standard kidney stone 24 hour urine includes measurements of volume, calcium, oxalate, phosphate, citrate, pH, sodium, potassium, magnesium, sulfate, ammonium ion and uric acid. Of these, three correlated with plaque abundance: urine volume, calcium, and pH. Plaque abundance varied inversely with volume and pH, and directly with calcium excretion.
Plaque abundance spans a nearly 100 fold range, thence we log transformed the vertical axis. In a general sense as a score made up from urine calcium, pH, and volume rises so does plaque abundance. The score is an arithmetic derived from multivariable linear regression. Each of the three components had an independent relationship to plaque.
Association, you might say, gives little clue as to cause. We agree. But its lack tells of near certainty in a negative way. So these associations moved us forward into a possible way that by its effects on urine calcium, volume, and pH kidneys might produce the plaque we so tediously measured. This work, linking renal physiology to plaque genesis remains for another article.
One should pause here and remind ourselves that Stoller, long before us, correlated plaque with hypercalciuria. That he did raises our confidence in the association.
Asked If Plaque Correlates with Stone Numbers
If stones form on plaque then more plaque should, or could, mean more stones. In our initial series of 15 patients, this proved true. Counting stones depends on both what radiographs show, what patients tell you, and what you can glean from hospital records.
Despite the complexity of counting new stones and measuring plaque, the two did correlate as this simple graph illustrates.
The four points at 0 stones come from four control subjects who did not form stones. The ‘plaque time score’ arose just like the prior score, from a multivariable linear regression analysis of factors that correlated with stones. Clearly time matters a lot, but so does plaque abundance.
Attached Stones Specifically Attach to Plaque
A glance at the prior figure will tell you that although plaque varies over a huge range, it occupies only between 1 – 10% of the papilla surface in most stone formers. This fact offered an opportunity for a rigorous test of the idea that it is indeed plaque itself that underlies and supports growth of stones on papilla.
Such a test took the form of a trial in which attached stones are graded as attached to plaque or not both during surgery and at confirmatory views of movies made during surgery. The trial used a fixed-sample testing and group sequential design to limit the numbers of patients needed. We terminated the study at 115 stones in 9 patients. By then, we had found 81 of 90 attached stones attached to plaque and calculated that more measurements could not affect the final outcome of the trial. We calculated the probability that 75% (95% CI 58 – 93%) of attached stones would be attached in replicate studies. This despite that plaque occupies only a minor fraction of papilla surface.
The Unattached Stones Had Grown on Plaque
You might have noticed that of 115 stones we accounted for 90. What about the other 25?
Of these, 12 showed precisely what Randall had found. They had on their surfaces unmistakable plaque remnants. Four were lost. That leaves nine to consider.
Micro -CT analysis scans each stone using x ray and reconstructs their internal regions by opacity to the radiation quantified exactly. Such radiographic density accurately identifies the various stone minerals. The remaining 9 stones thus analyzed all possessed internal apatite inclusions. We assumed they had detached and grown new layers of mineral to cover the old attachment site thus internalizing it. Not so exacting as much else we did, this work, but adequate to the concern at hand.
How Stones Form On Plaque
Randall knew calcium oxalate crystals grew over a plaque surface exposed to urine by failure of the overlying epithelial covering. But he could not determine what crystals and organic molecules formed the junction between stone and plaque. We helped define these. The original paper deserves reading by those with technical background. Multiple articles on this site detail plaque formation.
One is very detailed, the other presents overgrowth as part of a larger review. The third narrates the process in video form. None, however, show two rather dramatic pictures of the junction I much love.
The Stone Plaque Junction – From 30,000 feet
Here, in a biopsy from a patient, high resolution micro CT imaging shows a stone growing on plaque. Arrows outline the attached stone. Kidney tissue is pale because transparent to radiation compared to the calcium in the stone. Inside the tissue plaque fills considerable space, and a rising column of it meets the stone and forms the stone’s supporting pillar (double arrowheads).
Randall would have so enjoyed this remarkable image.
The Stone Plaque Junction – From a few microns
Here is a high resolution electron micrograph of the stone plaque junction.
The letter ‘B’ marks a protuberant fold of plaque in kidney tissue exposed to the urine. Black stained organic molecules from urine and perhaps tissue have coated the surface where the arrow points. The body of plaque in kidney tissue lies to the bottom left, the body of the stone to the upper right.
Inside the outermost black coating that faces onto the stone base, layers of crystals – white – and organic material – black alternate. That inside organic material is from the kidney, not the urine, and echos the layers of crystal and organic materials that make up the primary spheres of plaque we showed you just a few pictures back.
The single arrow to the right of the ridge labeled ‘B’ points to large rather edgy crystal plates that belong to the stone. The plates rest on and join the organic covering of the plaque that rides up between plates to form black spikes – one spike points to the top of the arrowhead.
The blending of urine crystals onto coated plaque is seamless and minute. That outer coating, some possible amalgam of urine and tissue proteins somehow interacts with the crystals, but in ways we do not know.
Once fixed to the papilla stone crystals proliferate looking a lot like ice floes in a freezing river. The double arrows and double arrowheads point to large crystal plates. Dark organic material shows through in places. But the main action has occurred. Anchored to plaque and bathed by supersaturated urine, the stone can grow at leisure until detachment.
Somewhere In Between
The studies at 1 micron like that above are so incredibly difficult technically as to virtually forbid the collection of multiple samples from different kidneys and patients. By contrast the micro – CT imaging of whole biopsies are facile but not highly resolved. This has led us to cultivate higher resolution micro-CT techniques such as this one.
In the intraoperative image of panel A, a less than 1 mm stone growing on plaque lies within the black circle at the arrowhead. The image also shows other scattered plaque, and a stone basket at 3 o’clock. We biopsied the tiny stone with its underlying tissue and made a high resolution CT image (B) rendered to show the outer surface of the stone (arrow) and underlying plaque (double arrows).
Image C shows the interior. Labels identify the plaque and overlying stone. Arrows point to collecting ducts with open lumens that pass through the calcified plaque. Smaller holes, without arrows, are thin limbs with open lumens. The radiographic textures of the stone and tissue plaque obviously differ. The arrowhead points to the interface between the plaque and stone.
Clearly one can use specific analytical methods to determine the crystals and perhaps organic molecules present in each area. Because it is less demanding than histological work with calcified tissue, this technique offers a way to study the stone plaque interface in multiple patients.
In the discussion ‘we’ referred to the research group, but Prof Jim Williams developed this method as an independent project. He did not publish this figure but gave us permission to use it here. In addition, he has reviewed our remarks and accepts them as correct.
The Organic Molecules
Unlike plaque itself, the organic molecules in stones and urine concerned many scientists since Randall, and our own work has been a scant fraction of the total. But if we narrow the focus to molecules associated with human plaque, knowledge is slim as yet.
Well known to bind to calcium crystals and retard their growth, OPN rises in kidney after diverse kinds of injury. Kidney stones contain a lot of it. We chose to study its relationship to plaque, and especially to its orientation toward plaque crystals.
Kidneys with plaque did not show higher than normal levels of OPN, but plaque seemed to accumulate it, perhaps via crystal binding. Cells of the inner medullary collecting ducts and thin limbs produced OPN.
As you may recall, plaque particles begin as spheres of alternating crystal and organic molecule laminations. At 35,000 fold magnification, OPN labeled with gold particles localizes in this plaque microsphere (arrows, picture to the right) within the organic layers. A layer of crystals lies between – white speckles – the organic layers.
At even higher magnification (left hand picture, 50,000 fold), OPN touches the surface of the crystal (white circle). All of the black dots of gold bound OP touch the crystal surface.
Such crystal orientation is what we would expect from a molecule well known for its ability to bind to crystals and affect their formation and growth.
Inter alpha Trypsin Inhibitor (IAT)
Why Study It?
In urine samples from many stone formers and non stone forming relatives Kristin Bergsland, one of our researchers, found this protein two of three whose levels correlated with stone formation. I say this because it is a compound protein made of three heavy chains (H1 – H3) and one light chain (bikunin) that can be linked in multiple combines. Bikunin (BK) and either the dimer of bikunin and H3 or the ITI trimer (BK + H1 + H2) correlated with stone forming.
Others had found that the combine molecule of BK and H3 (pre-α trypsin inhibitor, PαI) increased in animals given excess oxalate to promote stones, and in cells exposed to oxalate. Hyaluronan (HA), a complex sugar protein molecule, was known to bind H3 and other heavy chains to make complexes that stabilize the renal collagen matrix. This long chain of reasoning and prior measurements led us to look for HA and PαI in papillary biopsies. We expected to find it in and around plaque.
What Did We Find?
As always we studied biopsies from calcium oxalate stone formers with plaque. Normal tissue had almost no BK or H3 or HA, just OPN. The only exception was H3 and HA in the interstitium, that space between tubules and vessels. Not in the cells of the interstitium but between them, on the collagen.
The table says it all. D means the protein was diffuse, in the cell cytoplasm. A means it was just on the membranes facing onto the tubule fluid. M means it was in the organic layers of plaque particles. + means present, – means not present.
You can see the hot spots are loop cells. Remember plaque begins in the basement membranes of those cells. Likewise, the collecting ducts have them all. Plaque has H3 and, as we already know, OPN. By special double antibody staining OPN and H3 lay together, so called co-localized.
But in plaque microspheres, H3 was not like OPN. It did not localize to the mineral part but remained in the matrix – organic layers of the plaque. This means that whereas OPN may well get into plaque by binding to the mineral, H3 gets there as part of the organic materials that form around the mineral.
We made this image at 15,000 fold enlargement using ultra fast freezing to prevent certain kinds of artefacts. So the tissue outlines are not like those in other pictures in this article. But gold stained H3 (the prominent black line at the arrow in the central plaque particle) lay far from the white colored crystal in the middle.
What Did We Learn?
We worked very hard to do this work and even to write the paper. Given what we know, the H3 abundance may reflect attempts by kidney cells to stabilize the collagen around plaque. We think this because it operates that way in joint diseases where collagen is inflamed. We found no evidence of inflammation, but our tools are blunt enough we could pass it by unrecognized.
Bone Genes and Plaque
An obvious if subtle theory held that plaque arose from processes kin to bone. Presumably medullary interstitial cells differentiated into bone forming cells to do this work. We tested this idea and falsified it in one experiment. The occasion for the experiment was study of medullary sponge kidneys. Data from cell culture had suggested that bone genes were expressed and therefore that the many calcifications of MSK arose like bone does. To test this we measured two critical bone genes Osterix and Runx2 in tissues from MSK patients using immunostaining.
Indeed some cells expressed both genes. But none of the cells were in areas that contained any calcifications. On the other hand, many areas contained calcifications, yet no cells in those areas expressed the genes.
This led us to use the very same measurements in calcium oxalate stone formers with plaque. No cells from these patients expressed either gene even though plaque was abundant.
More or less these experiments falsified the bone gene plaque hypothesis.
We speak glibly about stones forming on plaque, breaking off, passing, and causing in this manner the miseries of stone disease. But what about the papilla? If stones break off plaque and take some plaque with them, and if plaque is embedded in the kidney tissue, does this not mean that successive stones may gradually wear away or abrade away the papillary tissue?
After long silence, we returned to plaque using endoscopic visualization and a reliable scoring system to answer this kind of question. We could score not only plaque abundance but also what we called pitting – a kind of trench where tissue has gone away, outlined by arrows. A bed of plaque lies just above it in brackets.
Unlike the feverish and minute work on molecules, or the groundbreaking discovery of where plaque originates, this work is leisurely statistical inference using fine surgical observations. Discovery, though limited by what we can measure has assured relevance.
Briefly, using standard associational tools, we found plaque abundance correlated with pitting. How many stones had passed, how many shock wave lithotripsies, both also correlated. Multivariable general linear modeling revealed that the primary association, adjusted for the rest, was between plaque and pitting.
This very conventional graph shows adjusted pitting scores plotted against increasing plaque scores. The p value for the trend upward is very low (<0.001) and therefore unlikely as a chance observation.
So we believe that more plaque may mean more stones that break off from plaque taking plaque and papilla tissue with them. As a result, papilla show pits where plaque was. Is this true? We do not know. It is an imagined view of what happens where we have no cameras going by day and night.
Molecules including OPN and IAT belong to a this ancient system that defends us against pathogens. Crystals resemble such pathogens whose outer coatings contain regular mosaics of surface charge such as bacteria and viruses. Kristin Bergsland wrote this useful review of the problem, but we have not pursued it experimentally. Many other so called crystal inhibitors belong to this same pathway, as she points out.
Our Plaque Work
What did we add to what Randall had left us?
Of course, its presumed site of origin. More, strong evidence that calcium oxalate stones grow on it, and a reasonable view of just how they do so. Rummaging about inside the tissues we have identified some likely molecules. Ranging into physiology we found, as Stoller before us, an association between plaque and urine calcium excretion. Perhaps more than anything we reignited interest in plaque and made clear that calcium oxalate stones in humans may well form without a necessity for crystals to adhere to tubule cells.
Why that last sentence?
Decades of work prior to us used animals given surplus oxalate. They plugged their tubules with calcium oxalate crystals. Stones clearly could grow over the open ends of those plugs bathed by the final urine. Massive researches disclosed details of cell injury, means of crystal attachment to cells, and the like. But in humans plugging of tubule cells with oxalate does not occur except under very uncommon and specific conditions such as primary hyperoxaluria.
Perhaps it is that we found no such plugs most benefited stone research. More, even, than our positive findings. Perhaps that, like Randall, we studied only human tissue and made progress – that fact alone – benefited others.
Plaque Work By Others
We have already mentioned the splendid work by Stoller before us, but want to review what others have done during our era. We exclude all work in rodents or with cells as being thus far not proven relevant to the common idiopathic human calcium oxalate kidney stone disease. Likewise we exclude all work having no original data but consisting in reviews or theoretical proposals. We have been thus stringent for ourselves, omitting all mention here of our many reviews.
Our search of PubMed for human plaque research was simply: randall[All Fields] AND (“plaque] OR (“plaque”[All Fields]. So it was highly inclusive. Because plaque links to amyloid formation, we specified exclusion of that term.
Using kidneys removed for cancer, Letavernier’s group performed a study of plaque localization. In other words they did not study stone formers but quite the contrary patients whose kidneys became available for other reasons altogether. They expected to find plaque in its earliest stages and, as good scientists often do, repeated much of what we did using this different tissue source. But with the addition of tubule segment specific antibody staining they could go further than us to resolve some issues of localization perhaps more perfectly.
Like us they found plaque at the papillary tips, with crystals of calcium phosphate. Plaque lay around thin limbs and vasa recta but almost never collecting ducts. They show microparticles that much resemble our plaque microspheres. They believe their observations most favored nucleation of the particles in the interstitium. Of most importance to the field, their findings match well with ours despite focus on nascent plaque in people without stones. Stones themselves and what causes stones could affect plaque. This work suggests the opposite.
What They Wanted to Know
I do not know who is the lead scientist in this group and therefore list it by first author. They harvested tissues from 7 normal people and 23 calcium oxalate stone formers. They biopsied papilla from the stone formers during PCNL. The ‘normal’ people had either ureteroscopy for cancer screening or nephrectomy for adhesion of adrenal tumor. The purpose was to discover gene expression differences related to plaque.
What Tissue they Used
This operative picture shows white plaque at the arrowheads and an obvious plug of a bellini duct at the arrow. Our article has avoided plugging, but others on this site give considerable background. Plugs, unlike plaque, produce obvious tubule cell death, and fibrosis and inflammation in the interstitium around plugged ducts.
The authors say they extracted RNA from whole papilla tissues to perform genome wide expression profiling. This means that they took whole biopsies, and measured differences between patients and normals in RNA abundances for all available genome elements. They noted that in some tissues plaque and plugs coexisted. Presumably, papilla from their ‘normals’ had no plaque or plugs. Although, the preceding paper points out that plaque can be found in exactly the kinds of patients this group took for their ‘normals’.
What They Found
In all they had data for 50,599 genes. They could compare papilla with evident plaque (and plugs) from stone formers to papilla with no evident plaque (plugs not stated) – from their ‘normals’.
As usual the result is an immense list of genes up or down regulated with – to us – no apparent relationship to how plaque might form or to stone disease. Inflammatory pathway genes seemed elevated. This makes us suspect that plugs affected some of their tissues. Plugs destroy lining cells and lead to obvious inflammation. The authors summarize their main findings in relation to renal injury, oxidative stress, and vasoconstriction.
No matter what one does with elaborate theorizing and speculation from a mass of nearly irreducible data, the basic tissue seems potentially faulty. If plugged tubules contaminated the so called plaque samples then they looked at the simple ravages of cell death from crystal nephropathy. Responsibility for proof otherwise lies with them.
We have said that stones often carry with them traces of the plaque on which they once had grown. This group found 10,282 of their 30,149 stones analyzed between 1989 and 2013 had such traces. Of interest to us the main mineral was calcium oxalate monohydrate in virtually all instances. I leave aside here issues with method as this group does know how to identify plaque. So we can take their results as certainty.
Their large series let them chart the fraction of all stones from the period noted against time, by sex. The results shows quite an increase over time, one they tallied as unlikely by chance. The three plots are 1989-1991 for the circles, 1999-2001 for the squares, and 2009-2011 for the triangles.
Among the younger patients, age 20 – 30 the fraction of plaque related stones rose very high indeed, to nearly 60 percent. By sheer area under the curves, men produced far more plaque related stones than women. Also, plaque wanes drastically with age.
They measured some possible vitamin d receptor gene variants by way of explanation but found nothing significant statistically.
To us, these results have many uses. Indeed time has altered things, and perhaps the reason why would enlighten us as to mechanisms of disease and improved treatment. But also, a large fraction of stones do seem to form on plaque.
Çiftçioglu et al
This person and colleagues attempted to identify a new class of calcifying nanoparticles with enough life like properties to warrant the name of nanobacteria. In collaboration with the Stoller group these investigators found evidence for nanobacteria in plaque harvested from human papillae. The results depend critically upon the specificity of antibody used for their detection. We found no subsequent confirmation of this work by others, and are inclined to a skeptical view until that occurs. Our group collaborated briefly with them and could not substantiate the validity of their antibody.
Mayo Clinic Group
John Lieske and Amy Krambeck have contributed massively to our knowledge about prevalence of plaque. I have already reviewed their work in another article, along with illustrations. They studied large groups of unselected stone formers and indeed found plaque as well as tubule plugging. But the latter far outweighed the former. Their large series was more predominantly female, whereas our high plaque group was the opposite. In a smaller series focused on idiopathic calcium oxalate stone formers, they described patients much like ours in having considerable plaque and being mainly younger men.
The French series reviewed above and the Mayo series can seem a bit at odds, in that they illustrate differing rates of plaque related stones – low in the large unselected Mayo Clinic group, large in the French study. But age may explain a lot. The French data show plaque related stones as a feature of youth, peaking in the 20 – 30 year old range, whereas the Mayo group age average approximated 60 or more. Sex also matters; women droop more at older ages, so that plaque stones are about 20% vs. 40% for men.
Integration Between Ourselves, Letavernier, Stoller, and Mayo
Science delights in offering different views of a single topic, a single problem. Where all concerned seem, as if by chance, to agree there one can build with great confidence.
All of us have found plaque, howsoever much we quibble about amounts. Likewise most stone patient studies associate plaque with calcium oxalate stones, male sex, youth – our main cases were younger males, and when measured hypercalciuria. By consensus, plaque lives in the papillary interstitium, at least in part. The French study of non stone formers make one believe that perhaps all kidneys can produce plaque albeit in amounts too small for detection without highly resolved imaging.
The marked fall in plaque related stones with age makes one wonder where it goes. If it builds up to form the nidus of stones in young men and women, do kidneys perhaps have ways to dispose of it? If one looks back on the clinical history of, let us say, the older women Lieske studied with Krambeck, who had scant plaque, did they form stones in their youth? Could one possibly track back actual stones produced over time in people asking if the frequency of plaque stones falls within individuals? Now that modern ureteroscopy permits high resolution images of papilla, perhaps repeated studies over time may show a waning of plaque in favor of other causes of stones – such as plugging.
Right now, one can say if high urine calcium fosters plaque then lowering urine calcium may reduce it. So proper stone prevention may well prevent plaque. Likewise, at least we found low urine volume as a factor, and all agree that high urine volume is good for preventing stones.
So be it. Time will bring more work, and we will add it here.