WHAT WE DO NOT KNOW ABOUT KIDNEY STONES
All of us know a great deal about stones, I mean the specific hard objects not the general field of stone research, but there is a void of majestic proportions and we all have encountered it. The crystals have been studied very well, and we know a lot about them. But the organic matrix has been very difficult to study and, from a clinical point of view, is totally inaccessible.
When I joined this field in 1969 we already knew that stones contained an obvious organic component. In my aging and well thumbed ‘Proceedings of the Renal Stone Symposium’ held at Leeds, April 1968 (J A Churchill Ltd, publishers, Standard Book Number 7000 1421 7) Professor WIlliam Boyce wrote a wonderful review of stone architecture that focused on the matrix. In it, he set the case forward in a ringing affirmation: ‘The best reason to consider calculous matrix is because it is there. In every human urinary concretion the matrix is present from centre to surface.’
The problem was, then, the sheer analytics; no techniques permitted resolution of matrix into its molecular components so real progress was stymied. In 2014 I reviewed what I found concerning the proteome of stones, and that review is below the present lead article by Witzmann. His work uses the most modern techniques and is a great advance over what has been. Below is my introduction followed by remarks by the author.
New data have been added, and this version replaces the prior one. Links have been changed and the old version is no longer in use.
Our own group, in particular Frank WItzmann and Jim Williams has added this work. Using the methods detailed in the paper, a total of 1,059 unique proteins could be identified in two human calcium oxalate kidney stones. Sheer prolixity, for want of a better word, of nature makes it hard to understand what all these proteins are doing in so modest a place. Probably many are irrelevant to stone formation and simply ride along with the crystals or even other proteins doing no useful work at all. But many are calcium binding and therefore prone to alter crystal dynamics. As a group the proteins in these two stones are known to have roles in the immune response system, inflammation, injury and tissue repair which raises the question of whether or not stones cause injury or inflammation or simply that these proteins, fitted to respond to invaders whose walls are generally highly charged are themselves anionic and prone to attach to crystals.
If you want to peruse the entirety here is our spreadsheet. There is also one with the paper itself.
I asked Frank to contribute remarks about his new paper, and here they are unedited as he wrote them:
Remarks by Frank Witzmann
“THE COMPLEXITY OF THE STONE MATRIX PROTEOME
Fred has posed a number of insightful questions:
‘You might think my writing self-contradictory to show you such an abundance of new molecular discoveries in stones under the title of what we do not know about kidney stones. And it is. We know more and more about the molecules. But we have almost no knowledge about how it all works. Are these stone matrix molecules simply surface active components of urine that adsorb to crystals as they form, no doubt affect how they form, and become part of stones? Are they in fact more than that: molecules that help bind crystals together to make clinically important stone mass? Are there data that the organic molecules ‘glue’ the crystals together? Being in immune and inflammatory pathways, are we to believe some of the molecules are a response to stones: does having stones set the stage for more stones by altering the urine proteome’?
Because of the previously successful application of our label-free quantitative mass spectrometry (LFQMS) platform in a broad range of biomedical questions, for which comprehensive differential protein expression analysis was needed, Jim Williams and I set out to use this approach to 1) improve and simplify the extraction of protein from kidney stone powder and 2) apply our novel, more comprehensive LFQMS approach to identify and quantify as many proteins as possible. We felt that previous attempts at this (which Fred has summarized) were inadequate for various technical reasons, and that a more comprehensive analysis might give us unique insights into stone formation. We have certainly achieved our immediate goals by identifying and comparatively quantifying 1,059 unique proteins in the organic matrices of two CaOx stones, classified as Ia and Id. As our paper concludes, we have corroborated and significantly expanded previous observations of the kidney stone matrix protein composition, and revealed a more complex matrix proteome than previously reported for human kidney stones of any type. But as Fred has already asked…”Do we have new testable hypotheses concerning how the matrix predisposes to stones?” The simple comparison of two CaOx stones may not be enough to answer that question, as it remains unclear as to whether the identified proteins, their physicochemical properties, and their associated pathways/functions are directly related to mechanisms of stone formation or simply coincidental through accumulation from long-term exposure of the growing stone to urine flow. For instance, we see some interesting differences between these stones, where only CaOx Ia contains neutrophil-associated proteins like calgranulin C, azurocidin, cathelicidin antimicrobial peptide, defensin 4, beta-defensin 1, neutrophil elastase, gelatinase-associated lipocalin, and lysozyme C; but CaOx-Id exclusively contains many kidney proteins like biglycan, dermicidin, destrin, napsin-A, osteoclast-stimulating factor 1, tight junction protein ZO-2, and uroplakin-3a.
Do proteins like these point to unique stone formation mechanisms?
Despite the limitations of our initial proteomic analysis, we can conclude that the comprehensive approach we have developed and reported here will enable us, and others who use it, to address more substantive questions about stone formation by analyzing and comparing comprehensive protein profiles in a broad range of stone types in relatively small stone specimens from individual patients who are carefully stratified by phenotype, and for whom important clinical data are known. We have recently completed just such a study in which well-characterized patient stone samples from male and female cohorts with CaOx and CaP (brushite) stones were compared using our LFQMS approach. The results are very interesting and it is clear that this kind of comparison may give us real insights into how the matrix is related to stone formation and which proteins are relevant to that process. Additionally, we have expanded our “stone proteome” database (to which Fred has provided a link) to include 1,721 different proteins. We will share our conclusions here when the CaOx – CaP comparison is published.”
Since these remarks are in addition to the paper itself, they are valuable reading as a supplement.
The papers below are ones I reviewed over a year ago and remain of interest.
Canales et al
Canales and his colleagues extracted the organic material from seven pure COM stones and found 68 proteins; of these 50 were considered by the authors as previously unidentified in stone matrix. Myeloperoxidase chain A (MPO-A), α defensin, and the the calgranulins (there are 3 found so far in stones) may be part of the immediate immune response. In addition they found three cell injury proteins, one stress response protein, an array of plasma proteins such as albumin, hemoglobin and transferrin, carbonic anhydrase, and a mixture of proteins that include osteopontin. Finally they found cell adhesion, membrane transport, biogenesis, cell signalling and coagulation proteins. The Tamm Horsfall protein was also found as most of us would expect.
Merchant et al
Merchant and colleagues did much the same kind of work. From stones of 5 people not otherwise described they identified 58 proteins, of which 11 were high abundance and 10 of these present in all three analyses of the extracts from stones. Of these, all but 1 had been previously been identified as binding to CaOx or HA surfaces. Four proteins were prevalent and high abundance on the basis of normalized abundance computation: calgranulins A and B, apolipoprotein A-1, and THP. Using ingenuity analysis, 3 principle pathways were identified: tumorigenesis, immune, and inflammation. The top canonical pathway was acute phase response signaling. The serious weakness of this work is in the stones: unknown crystals.
Okumura et al
Okumura et al obtained stones from 9 patients otherwise unidentified. All were predominant CaOx by FTIR. They noted electrophoretic mobilities of certain matrix proteins, such as THP were very heterogeneous and decided to do in solution protease digestion followed by proteomic analysis. This differs from most other studies which did peptide digestion and analysis from bands of gels.
All stones had some osteopontin, prothrombin F1 fragments, THP, calgranulins A, B and C, myeloperoxidase, and albumin (See Featured figure at the top of this post). What is striking is the variation of protein abundances among the stones. The PTF1 fragment abundance varied over 10 fold; for THP variations were slighter. Calgranulins A and B were almost inverse to PTF1 but ran together.
This variability points to where I think new research can profitably be done. Some of the variability surely arises from heredity. It may be part of the well known heritability of stones themselves. Possibly it is in part a reflection of the effects of stones or of the tissue injuries from procedures used to treat stones.
This post is selected from a longer review which is public but can be reached only through this link. I have made no effort to pretty it up but the links are correct and may be of use to you. I may keep it current if comments come back indicating that would be a good idea. If I have missed important references, perhaps yours, please let me know.
You might think my writing self contradictory to show you such an abundance of new molecular discoveries in stones under the title of what we do not know about kidney stones. And it is. We know more and more about the molecules. But we have almost no knowledge about how it all works. Are these stone matrix molecules simply surface active components of urine that adsorb to crystals as they form, no doubt affect how they form, and become part of stones? Are they in fact more than that, molecules that help bind crystals together to make clinically important stone mass? Are there data that the organic molecules ‘glue’ the crystals together? Being in immune and inflammatory pathways, are we to believe some of the molecules are a response to stones: does having stones set the stage for more stones by altering the urine proteome?
We have far better tools than in 1969. Do we have new testable hypotheses concerning how the matrix predisposes to stones?