WHAT WE DO NOT KNOW ABOUT KIDNEY STONES
Kidney stone matrix proteins glue kidney stone crystals together to make the stones we encounter and treat. Some of the matrix proteins control whether kidney stone crystals form at all, and the rates that they can grow at. Some call these ‘inhibitors’ or ‘promoters of kidney stones, although their exact role in kidney stone formation remains uncertain to today. Many matrix proteins belong to the immune system, or the inflammatory response.
Our Lack of Knowledge
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 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.’
Limitations of Analytical Methods
The sheer analytics styme us. No techniques until now have permitted resolution of matrix into its molecular components. In 2014 I reviewed what I found concerning the proteome of stones, and that review lies below the present lead article by Witzmann. His work uses the most modern techniques and is a great advance over what has been.
The Main Results
Using the methods detailed in the paper, we identified a total of 1,059 unique proteins in two human calcium oxalate kidney stones.
Sheer prolixity, for want of a better word, of nature makes it hard to understand what these proteins do. 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 could 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.
Comments By Frank WItzmann
A New Method
Because our label-free quantitative mass spectrometry (LFQMS) platform has performed well in the past, we used it to extract protein from kidney stone powder and identify and quantify as many proteins as possible. Since previous attempts seemed inadequate for technical reasons, we thought our more comprehensive analysis might give us unique insights into stone formation.
We have quantified 1,059 unique proteins in the organic matrices of two CaOx stones. They corroborate and expand on previous observations of the kidney stone matrix protein composition. As well, they reveal a more complex matrix proteome than previously reported for human kidney stones of any type. But do we have new testable hypotheses concerning how the matrix predisposes to stones?
Does it Matter?
The simple comparison of two CaOx stones may not be enough to answer that question. The two differ markedly. Only one contains neutrophil-associated proteins like calgranulin C, azurocidin, cathelicidin antimicrobial peptide, defensin 4, beta-defensin 1, neutrophil elastase, gelatinase-associated lipocalin, and lysozyme C. Only the other contains 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 believe our approach will enable us to analyse and compare comprehensive protein profiles in small stone specimens from individual patients. We have already studies kidney stone samples from male and female cohorts with CaOx and CaP (brushite) stones. We will share our conclusions here after the CaOx – CaP comparison is published.
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 the authors considered 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. They found Tamm Horsfall protein 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. Because they found very heterogeneous electrophoretic mobilities of certain matrix proteins, such as THP, they 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 of the 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 itself suggests profitable research. Some of the variability surely arises from heredity. It may cause part of the well known heritability of stones themselves. Possibly variability reflects the effects of stones or of the tissue injuries from procedures used to treat stones.
I redacted this article from a longer review you can reach through this link. Although I made no effort to pretty it up, you may enjoy it. If I have missed important references, perhaps yours, please let me know.
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? Do they affect how crystals form, and become part of stones? Do they bind crystals together to make clinically important stone mass? Being in immune and inflammatory pathways, are some of the molecules 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?