Introduction

Over the past 50 years or so, much has been written on the topic of inhibitors of the crystallisation of calcium salts in urine claiming that they may protect the individual against the formation of calcium-containing kidney stones. The wide range of candidates for this role includes magnesium [1], citrate [2], pyrophosphate [3, 4] adenosine diphosphate [5], adenosine triphosphate [5], two phosphopeptides [6], various glycosaminoglycans [7, 8], non-polymerised Tamm–Horsfall protein (also known as uromodulin) [9, 10], nephrocalcin (now known to be the same as bikunin) [11–14], albumin [15], osteopontin (also known as uropontin) [16, 17], calgranulin [18], α-1-microglobulin [19], β-2-microglobulin [20], matrix Gla protein [21], urinary prothrombin fragment 1 [22, 23], human urinary trefoil factor 1 (THF1) [24] and inter-α-trypsin inhibitor (bikunin light chain) [25, 26]. Many of the macromolecules in this list appear to act by binding to calcium sites on growing crystals through polyanionic sites such as the gamma-carboxyglutamic (GLA) domain in their protein structure. Of the above list, only citrate and magnesium have been found to be excreted in lower amounts by some calcium stone-formers than by normal subjects [27]. There have also been occasional claims that stone-formers may excrete lower amounts than normal subjects of some of the macromolecular members of the above list [28]. However, the excretion of none of these ions and macromolecules has been found to discriminate exclusively between stone-formers and normal subjects.

Other urinary constituents have been identified that are claimed to be promoters of the crystallisation process. These include matrix substance A [29, 30], various uncharacterized urinary proteins and glycoproteins [31–33], polymerised Tamm–Horsfall protein (also known as uromucoid) [34, 35], albumin [15, 36, 37], lysozyme [38] and lactoferrin [38]. For a comprehensive review of the various constituents in urine which have been claimed to inhibit or promote the formation of calcium-containing stones in the urinary tract, the reader is referred to an excellent article by Aggarwal et al. [39] and to other articles in this Special Issue [40, 41] which deal in more detail with the identification of individual urinary inhibitors and promoters of crystallisation and their claimed effect on the crystallisation of calcium salts in urine. Instead, this current article will attempt to answer the more general, but nevertheless important, question—do any of these so-called “inhibitors of crystallisation” actually play a role in the prevention of stone-formation based on evidence contained in the literature and in previously unpublished data from a number of laboratory records?

Originally, many of the above urinary constituents were shown to modify one or more of the steps involved in the crystallisation of both of these salts solely on the basis of in vitro studies. These test systems generally employed relatively simple supersaturated solutions which bore little or no resemblance to the overall composition of urine [1–9]. Eventually, test systems were devised which utilised more complex multi-component solutions, such as those used in constant composition [42, 43], mixed-suspension-mixed-product-removal (MSMPR) (the latter sometimes referred to as continuous crystallisers) [35, 44–47] and reverse osmosis systems [48]. It was then assumed that these inhibitory factors played a similar role in “whole” urine. I will be the first to admit that I originally fell into this trap myself [7] but have since realised that there are many more factors to be considered before it can be accepted that these apparent in vitro “inhibitors” actually play a role in stone prevention. Subsequent studies have been carried out on the effects of some of these substances in more “urine-like” solutions [44–48] and even in urine itself [48, 49] but there are many problems associated with both the theory behind the test systems employed and the practical aspects of the tests themselves.

It has been claimed that the various so-called “inhibitors of crystallisation” identified using the above test systems can act on a number of processes in the overall course of crystallisation. These include (1) inhibition of what may be termed “crystal self-nucleation”,Footnote 1 (2) inhibition of crystal growth, (3) inhibition of crystal aggregation, (4) inhibition of the binding of calcium-containing crystals to tubular cell epithelium or (5) inhibition of crystal growth on Randall’s Plaques or Randall’s Plugs.

But what is the evidence then that so-called “inhibitors of crystallisation” play any role in inhibiting one or more of the above steps in the crystallisation process or in preventing the formation of stones in the urinary tract? Since there are few, if any, data in the literature on urinary inhibitors of the crystallisation of cystine, uric acid or magnesium ammonium phosphate, the remainder of this article will concern itself with the various ions and macromolecules which have been proposed to play a role in preventing the formation of stones consisting of either calcium oxalate (CaOx) or calcium phosphate (CaP) or both.

Inhibitors of crystallisation of calcium salts

As mentioned above, there are three main steps in the crystallisation of calcium salts in urine plus two other mechanisms by which crystallisation might take place where the so-called inhibitors might play a role in delaying or blocking the process. Let us look at the concepts involved in these various stages and the evidence that these so-called inhibitors play any role in modifying them:

Inhibitors of spontaneous “self-nucleation”

Since the late 1960s, many studies have been carried out on so-called “inhibitors of spontaneous self-nucleation”. Most have been concerned with measuring the effect of these substances on some empirically determined upper limit of supersaturation at which crystal nucleation appears to take place, sometimes referred to as the formation product (FP) or metastable limit (ML) or upper limit of metastability (ULM) of the calcium salt concerned. The problem is that many of the studies were carried out under conditions incompatible with those involved during the elaboration of urine in the renal tubules. Most importantly, the majority employed incubation times which were quite unrealistic when compared with the actual transit time of urine through the renal tubules (about 3–4 min). The importance of time of incubation in determining whether or not a given so-called “inhibitor of self-nucleation” is relevant to the initiation of stones within the transit time of urine through the renal tubules can be seen from the studies in Figs. 1, 2, 3, 4 and 5, which show the effect of time of incubation and inhibitor concentration on the nucleation of crystallisation of CaOx and CaP in simple metastable solutions (Figs. 1, 2, 3, 4) and in urine (Fig. 5).

Fig. 1
figure 1

The effect of time of incubation on the formation product of calcium oxalate (CaOx) from various studies compared with the 4-min formation product of CaOx determined by Robertson [50]

Full size image
Fig. 2
figure 2

The effect of time of incubation on the formation product of calcium phosphate (CaP) compared with the 4-min formation product of CaP determined by Robertson [50, 51]

Full size image
Fig. 3
figure 3

The effect of increasing concentrations of pyrophosphate on the 20-h formation product of CaOx as determined by Fleisch and Bisaz [4] compared with the 4-min formation product of CaOx of Robertson [50]

Full size image
Fig. 4
figure 4

The effect of increasing concentrations of various diphosphoric acid salts on the 3-day formation product of CaP as determined by Fleisch and Bisaz [3] compared with the 4-min formation product of CaP of Robertson [50]

Full size image
Fig. 5
figure 5

The original RSS of CaOx and the 4-min formation product of CaOx measured in urine samples from six normal subjects calculated using SUPERSAT [57] in relation to the 4-min formation product of CaOx as determined by Robertson [50]

Full size image

First, and most importantly, the FP/ML/ULM values for both CaOx and CaP in simple metastable solutions are time-dependent (Figs. 1, 2) i.e. the longer one leaves a given metastable solution, the more likely it is to crystallise out. This is simple school chemistry! The Figures include data from my own PhD studies [50, 51] but also data from other workers in the field who have determined FP/ML/ULM at various incubation times ranging from a few hours to 3 days or even longer [3, 4, 52–54]. These contrast with the very short-time FP/ML/ULM values for these salts obtained by myself when I attempted to simulate the supersaturation conditions that would be necessary to cause the “self-nucleation” of CaOx and CaP within the transit time of urine through the nephron (3–4 min) [50, 51]. For these short-term studies, CaOx was nucleated by titration of metastable solutions of CaOx with sodium oxalate and CaP nucleated by titration of metastable solutions of CaP with sodium hydroxide. The data from these studies show that the supersaturation levels required to initiate the crystallisation of these salts within 3–4 min are much higher than those required at the 6-, 20-h and 3-day incubation times variously employed by other workers in the field [3, 4, 52–54]. Since urine rarely, if ever, remains in the renal tubules or even in the renal calyces for such long times of incubation, the FP/ML/ULM values obtained at these relatively long time periods would seem to be totally irrelevant to the stone initiation process and, perhaps, should be disregarded in any consideration of the “free-particle” model of stone-initiation. It might be argued that incubation times up to a few hours might be relevant to the initiation of the crystallisation of calcium salts in the bladder but, as far as the more proximal sections of the urinary tract are concerned, they would only apply to that small number of cases who develop urinary stasis in the renal pelvis and/or ureter caused by some anatomical abnormality.

The second piece of evidence for questioning the early claim for the existence of so-called “inhibitors of self-nucleation” is shown in Figs. 3 and 4. These show the effects of pyrophosphate and other modifiers of crystallisation on the FP/ML/ULM of CaOx and CaP at time intervals ranging between 6 and 24 h or more [3, 4, 55, 56]. These are shown in relation to the 3–4 min FP values for these salts in the absence of inhibitors as determined by Robertson [50]. This shows that the maximum effect of these inhibitors on the FP/ML/ULM of these salts at 20–24 h only takes the FP/ML/ULM value up to the 3–4 min FP values of Robertson but never exceeds them. This suggests that any potential effect of these inhibitors on spontaneous “self-nucleation” at periods longer than 3–4 min can be overwhelmed if the supersaturation of urine reaches or exceeds the 3–4 min FP value of the salt concerned. In other words, these so-called inhibitors of “self-nucleation” become ineffective at the levels of supersaturation that lead to spontaneous rapid nucleation of calcium salts in urine.

This hypothesis is further supported by the data in Fig. 5 which shows the results of short-term studies on CaOx nucleation in whole urine which had been freshly collected from normal subjects and filtered through a 2 µm grade filter to remove any pre-existing particulate material [50]. Presumably, these urine samples still contained most, if not all, of the above inhibitors claimed to be of importance in the prevention of crystal nucleation. Figure 5 shows that the 3–4 min FP/ML/ULM of CaOx in these urines is close to that found in simple inorganic solutions, once the actual supersaturation of urine is taken into account. The supersaturation levels of the urine samples were calculated using the updated version of SUPERSAT program [57] (an early predecessor of EQUIL 2 [58]). This supports the above suggestion that any inhibitors that were present in the urine, including all of those mentioned in “Introduction”, do not have the ability to increase the 3–4 min FP/ML/ULM of CaOx, once the potential complexation of calcium and oxalate to these constituents has been taken into account in the calculation of supersaturation. Therefore, in terms of short-term (i.e. 3–4 min) “self-nucleation” of calcium salts in urine, supersaturation is by far the dominant factor in the process and inhibitors appear to play little, if any, part in delaying the onset of nucleation at these high levels of supersaturation.

Figures 3, 4 and 5 add further support to the overall hypothesis that studies purporting to show that certain constituents of urine can inhibit the “self-nucleation” of calcium salts are irrelevant because they were either carried out using unrealistic incubation times or did not take into account the fact that supersaturation may overwhelm any potential inhibitory effect of these compounds. The conclusion from these observations is that claims that these compounds (a) affect the upper limit of metastability or the metastable limit of calcium salts in urine and (b) are important in preventing the initiation of stones appear to be unsupported.

The important question then remaining is—does tubular fluid ever actually reach the 3–4 min FP values for CaOx and CaP within its transit time through the nephron? Recent calculations using a computer model of tubular fluid composition as it progresses through the nephron have shown that this is possible following transient increases either in plasma oxalate, e.g. after ingestion of a meal high in oxalate (see on “Inhibitors of crystal aggregation” below) in the case of calcium oxalate crystallisation, or following a small increase in either plasma phosphate or plasma pH in the case of calcium phosphate crystallisation [59].

If inhibitors are unlikely to influence the “self-nucleation” of calcium salts within the transit time of urine through the nephron, is it possible that they may have an effect on the subsequent crystal growth and/or agglomeration of crystal nuclei once formed? The answer to this may be “Yes” but, as will be shown below, excessive supersaturation can frequently overwhelm the effect of these inhibitors at least on the crystal growth phase.

Inhibitors of crystal growth

Many studies have shown that there are numerous constituents of urine which have the ability to inhibit the rate of growth of CaOx and/or CaP crystals in vitro [2, 5] although many of the early studies were carried out using simple solutions which only faintly resembled the actual composition of urine. As mentioned above, such studies have been criticised as possibly being irrelevant to the stone-forming process since many of the so-called inhibitors used could be influenced by some of the many urinary factors omitted from the test systems employed. There are, however, some in vivo studies which claim that some of these inhibitors may have an effect on the crystal growth of calcium salts in the urine of stone patients and, in some instances, may actually lead to a reduction in stone recurrence. Principal among these are potassium citrate medication and, to a lesser extent, magnesium supplements, which have been claimed to reduce stone recurrence by increasing urinary citrate excretion [60–62] and magnesium [63–65], respectively. I have to say, however, the claimed beneficial effect of potassium citrate medication did not materialise in our study carried out in a small group of patients with recurrent CaOx stones treated in this way (Unwin and Robertson, unpublished data) and was not effective in small doses in another study [66]. In our study, potassium citrate did not increase urinary citrate and the patients were “converted” from being “pure” CaOx stone-formers to being “mixed” CaOx + CaP stone-formers as a result of the alkalinisation of their urine, a potential risk that was pointed out by Coe and his colleagues [67] and recently confirmed in a rat model by Krieger et al. [68]. Our patients continued to form stones at the same or even higher rate than before. Even if administration of citrate (either as potassium citrate or potassium magnesium citrate), does have a beneficial effect on stone recurrence in larger groups of patients, as claimed by several groups [60–62], the potential for both citrate and magnesium to reduce the level of supersaturation of urine with respect to CaOx and CaP (as a result of their ability to complex with calcium, in the case of citrate, and oxalate and phosphate, in the case of magnesium) cannot be ignored. In addition, the patients studied on these treatment modalities were frequently also advised to drink more water which would further decrease the level of supersaturation with respect to both calcium salts. So, it is difficult to disentangle the precise mechanism(s) by which these treatments may have actually worked. Given the predominance of supersaturation over inhibitory activity as a determinant of crystal nucleation, as suggested in “Introduction”, it might be that the claimed beneficial effects of potassium citrate and magnesium potassium citrate on stone recurrence rates are more related to their ability to reduce supersaturation rather than to any increase induced in the inhibitory activity of the urine concerned.

One of the earliest naturally occurring urinary inhibitors of crystallisation to be identified was pyrophosphate, the properties of which were extensively studied by Fleisch and Russell and their colleagues in Switzerland [3, 4, 69]. From the early 1960s onwards, this group conducted many in vitro studies on the inhibitory effect of pyrophosphate on the crystallisation of both CaOx and CaP as shown in Figs. 3 and 4 [3, 4]. Their findings were later supported by the results obtained from more sophisticated studies on the effects of pyrophosphate and other polyphosphates on the crystal growth of CaOx carried out by Meyer and Smith at the Mayo Clinic [2, 5]. Their in vitro studies on pyrophosphate led the Mayo Group to treat CaOx stone-formers with phosphate supplements, which are known to increase the urinary excretion of pyrophosphate by inhibiting its tubular reabsorption through competition for a common reabsorption site in the renal tubule [70]. Studies followed which showed that phosphate supplements could reduce the rate of recurrence of stone-formation in CaOx stone-formers [71–73]. However, yet again there was a dual effect of this form of treatment on urine composition since the high doses of phosphate not only increased urinary pyrophosphate excretion but also reduced the intestinal absorption of calcium by precipitating it as calcium phosphate, thereby leading to a decrease in urinary calcium and the relative supersaturation of urine with respect to CaOx. So, it is not clear that the observed increase in urinary pyrophosphate was the main or even partial cause of the observed decrease in stone recurrence. Subsequently, phosphate supplements fell out of favour as a treatment for idiopathic calcium stone-formers because of fears that the potential fall in plasma calcium, following the reduction in the intestinal absorption of calcium, might stimulate the parathyroids. However, in spite of this potential side-effect, phosphate supplements are still used in some patients with primary hyperoxaluria [74] for whom there is no alternative completely satisfactory form of prophylaxis other than a very high fluid intake or, in about 21% of cases in whom it is effective, pyridoxine supplements [75].

In an early attempt to use synthetic inhibitors of the crystallisation of CaOx to reduce stone recurrence in highly recurrent idiopathic CaOx stone-formers and based on the findings from in vitro and animal studies [76, 77] carried out by Fleisch’s group, the Leeds group gave an oral dose of ethane-1-hydroxy-1,1-diphosphonate (EHDP) for 4 weeks to a small group of such patients in order to try and reduce CaOx crystalluria by inhibiting the crystal growth and/or crystal aggregation of that salt [78]. EHDP, now known as 1-hydroxyethane-1,1-diphosphonate (HEDP) or etidronate, was one of the early bisphosphonates which was originally used in studies aimed at reducing bone resorption but which has now been superseded by more effective bisphosphonates with fewer side-effects, such as alendronate, ibandronate, risedronate and zoledronate [79]. Because EHDP could only be given orally at the time of our studies and because it is poorly absorbed by the intestine, it had to be prescribed in relatively high doses (80 µmol/kg body weight/day). Unfortunately, at this dose level it appeared to precipitate or complex with sufficient calcium in the intestine (a) to reduce the urinary excretion of calcium and (b) to free up oxalate within the contents of the intestine (oxalate which would have otherwise been precipitated with the calcium now bound to EHDP) thereby rendering this oxalate available for passive absorption in the large bowel. As a result, urinary oxalate increased by over 50% which, in turn, caused CaOx crystalluria to increase markedly, both in terms of amount and average particle size, when compared with the crystalluria observed in the same patients on their baseline diets (Fig. 6). Although many of the crystals and aggregates of CaOx were smaller than those found in the fresh, warm urines from the same group of stone-formers given a small dose of sodium oxalate to simulate the hyperoxaluric effect of the EHDP, a significant number of very large crystals and aggregates of CaOx were still frequently observed (Figs. 7, 8). The data were interpreted to show that, although for part of the time EHDP seemed to be excreted in sufficient quantities to inhibit the growth and aggregation of CaOx crystals, there were other occasions when the supersaturation of urine with respect to CaOx seemed to overwhelm the inhibitory effect of EHDP on at least on crystal growth. The data also confirmed the conclusion drawn from the in vitro studies shown in Fig. 3 that EHDP could not inhibit the initiation of CaOx crystallisation at the high levels of supersaturation observed in vivo and was not, therefore, an inhibitor of “self-nucleation” under these conditions.

Fig. 6
figure 6

The total CaOx crystalluria measured in the fresh, warm urine samples from recurrent CaOx stone-formers a on their basal diet, b on their basal diet augmented by a single dose of sodium oxalate (2.6 mmol) given before breakfast and c on their basal diet augmented by a daily dose of EHDP (80 µmol/kg body weight/day). (Dashed line upper limit of normal)

Full size image
Fig. 7
figure 7

The percentage of large crystals and aggregates of CaOx measured in the fresh, warm urine samples from recurrent CaOx stone-formers a on their basal diet, b on their basal diet augmented by a single oral dose of sodium oxalate (2.6 mmol) given before breakfast and c on their basal diet augmented by a daily dose of EHDP (80 µmol/kg body weight/day). (Dashed line upper limit of normal)

Full size image
Fig. 8
figure 8

Photomicrographs of CaOx crystals and aggregates measured in the fresh, warm urine samples from recurrent CaOx stone-formers a on their basal diet, b on their basal diet augmented by a single oral dose of sodium oxalate (2.6 mmol) given before breakfast and c on their basal diet augmented by a daily dose of EHDP (80 µmol/kg body weight/day)

Full size image

More recently, in vitro studies on the crystallisation of CaOx using a continuous crystalliser, have shown that at least three of the above more sophisticated bisphosphonates currently used in the treatment of osteoporosis [79], slightly reduced the crystal growth of the CaOx crystals produced but had a major inhibitory effect on the aggregation of these crystals [80]. Yet again, the data confirmed that these bisphosphonates could not inhibit the spontaneous “self-nucleation” of CaOx crystallisation at the high levels of supersaturation employed in the continuous crystalliser and did not appear, therefore, to be inhibitors of “self-nucleation”. Since these bisphosphonates were shown to be effective at micromolar concentrations (concentrations which are estimated to be reproducible in urine following treatment with these forms of medication), it may be that one or more of these compounds may eventually be useful for preventing the formation of CaOx stones, a possibility supported by studies in rats [81]. Of course, the treatment would have to be limited to adults since bisphosphonates could potentially interfere with the development of bone in children.

Inhibitors of crystal aggregation

Attention was first drawn to the potential role of crystal aggregation as a risk factor for stones in the late 1960s [82, 83] and this led to many subsequent quantitative studies on the possible role of crystalluria and particle size in the initiation of stones [84–86]. As a result of these studies, many workers, although not all, became convinced that crystal aggregation is a vital step in the initiation and continued growth of stones [87, 88]. Since crystal growth is a relatively slow process, it was readily appreciated that the most rapid way to increase the particle size of incipient crystals of calcium salts was through crystal aggregation and not through crystal growth per se. The importance of crystal aggregation was given further support when studies with fresh, warm urine from recurrent CaOx stone-formers and normal subjects showed that the urine from patients often contained large crystals and aggregates of CaOx whereas that from normal subjects never contained such large particles although they occasionally passed small individual crystals of CaOx when their urine exceeded the 3–4 min FP of that salt. Figure 9 shows the percentage of large crystals and aggregates in the fresh, warm urine samples passed by a group of recurrent idiopathic CaOx stone-formers and in the urines of normal subjects in relation to the level of supersaturation of urine with respect to CaOx. This shows that, as mentioned above, the stone-formers passed much larger crystals and aggregates of CaOx than did the normal subjects at all levels of supersaturation above the 3–4 min FP of CaOx. These data were originally interpreted to show that the stone-formers had less inhibitory activity towards the growth and aggregation of CaOx than did the normal subjects and this led myself and many others to study inhibitors (and also promoters) of crystallisation for several years. Many urinary constituents were shown to inhibit not only the crystal growth of CaOx and CaP, as mentioned above, but also, more importantly, the aggregation of these crystals. As a result, a deficiency in the urinary excretion of inhibitors of growth and aggregation by stone-formers compared with normal subjects was suggested to be the cause of the difference in crystalluria patterns between the two groups [85, 86].

Fig. 9
figure 9

The percentage of large crystals and aggregates of CaOx measured in the fresh, warm urine samples from recurrent CaOx stone-formers (RSF) and normal subjects (N) in relation to the relative supersaturation (RSS) of urine with respect to CaOx. The data are shown in relation to the Solubility Product (SP) and 4-min formation product (FP) of CaOx [50]

Full size image

More recently, however, I have come to an alternative possible interpretation of the data in Fig. 9. A second explanation for the difference between the CaOx crystal sizes excreted by normals and recurrent stone-formers emerges from a computer model of the functioning of the renal tubule, which shows that there are two possible points in the nephron where CaOx crystallisation may be initiated, particularly in the tubular fluid of recurrent stone-formers [59]. The first occurs at the end of the descending limb of the loop of Henle (DLH), following a small transient increase in plasma oxalate and secretion of oxalate into the lumen towards the end of the proximal tubule. The second occurs in the latter part of the collecting duct (CD) where supersaturation increases for a second time following the final adjustment of water reabsorption which occurs throughout the CD section of the tubule. Those crystals initiated at the end of the DLH by spontaneous “self-nucleation” may be delayed in transit through the renal tubule by various physical and hydrodynamic mechanisms described in the paper [59] and grow into the large crystals and aggregates frequently observed in fresh, warm urine collected from recurrent stone-formers. In contrast, the model shows that those crystals initiated in the CD will not grow fast enough in the remaining transit time of fluid through the latter part of the CD and will be excreted as relatively small crystals measuring only a few microns in diameter, such as observed in the urines of many recurrent stone-formers and occasionally in the urines of some normal subjects [83]. Thus, the model predicts that normal urine should contain a unimodal peak of small crystals of CaOx and that the urine from recurrent stone-formers should show a bimodal distribution of particle size consisting of an initial peak of small crystals and a second of much larger crystals and aggregates of that salt.

These predictions from the model are supported by the findings from an in vivo study, carried out in the early 1970s, in which six idiopathic calcium recurrent stone-formers (RSF) and six normal control subjects (N) were fed a fixed basal diet for 4 days [84]. On the second day of the study, urine samples from both groups were collected into warm vacuum flasks at 09.00 (immediately before breakfast) and then at 3-h intervals over the following 12 h and finally at 09.00 the next morning. The size profiles of CaOx crystals and aggregates contained in the urine samples (maintained at 37°) were measured within 10 min of voiding using a Coulter Counter. The size and appearance of the crystals and aggregates were also checked by light microscopy immediately after voiding. On the fourth day of the study, a small oral dose of sodium oxalate (2.6 mmol—equivalent to the quantity contained in about 30 g of dry spinach or 40 g of boiled spinach) was administered to the patients and control subjects immediately before breakfast at 09.00 and fresh, warm urine samples collected according to the above protocol. Again, the size profiles of CaOx crystals in the urine samples were measured within 10 min of voiding using a Coulter Counter and the size and appearance of the crystals and aggregates of CaOx checked by light microscopy on a warmed microscope stage.

Figure 10 shows the mean size distributions of CaOx crystals and aggregates in the fresh, warm urine from the normals and the RSF at 12.00 on the second day of the basal diet and Fig. 11 shows the corresponding data on Day 4 of the study, 3 h after the sodium oxalate bolus was given. On the basal diet, both groups produced a peak of small CaOx crystals (mean diameter ~6 µm) in the urine samples taken at 12.00 although the peak in the urine from the RSF was much higher than that in the urines from the normals. However, the urines from the RSF exhibited a second peak of much larger CaOx crystals and aggregates (mean diameter ~25 µm, but some as large as 50 µm, which was the upper limit of measurement in the Coulter Counter system used at that time). In fact, the light microscopy studies showed that some of the particles were much larger than 50 µm and these particles frequently blocked the 65 µm aperture of the Coulter Counter probe. After the oxalate load, the normals showed an increase in the peak of small crystals of CaOx but still did not pass any large crystals or aggregates in their urines at that point or during the remainder of the day. The RSF, on the other hand, showed a marked increase in the peak of large crystals and aggregates and 3 out of the 6 patients had attacks of renal colic within a few hours following the oxalate load. Urinary oxalate excretion increased within the first 3 h in both groups but slightly more so in the RSF than in the normals. Furthermore, the normals had a slight diuresis, which helped to moderate the increase in oxalate concentration in their urine (0.22–0.28 mmol/l) caused by the ingestion of the sodium oxalate. The RSF, on the other hand, had little or no diuresis and their urinary oxalate concentrations increased from 0.30 mmol/l to 0.52 mmol/l. The reason for this difference in the diuretic response to the oxalate load between the two groups is not clear but may be worthwhile investigating. In this connection, it is interesting that dandelions, which are known in France as “pis-en-lit” (literally piss-in-the-bed), have a high content of oxalate and are often used as a diuretic in homeopathic medicine. Perhaps, some stone-formers have a reduced ability to exhibit this “protective” diuretic response to an oxalate load relative to that shown by normal subjects and this exacerbates their risk of forming CaOx-containing stones after a meal high in oxalate?

Fig. 10
figure 10

The volume of CaOx crystalluria in relation to particle size in the fresh, warm urine samples from recurrent CaOx stone-formers (RSF) and normal subjects (N) on a fixed basal diet

Full size image
Fig. 11
figure 11

The volume of CaOx crystalluria in relation to particle size in the fresh, warm urine samples from recurrent CaOx stone-formers (RSF) and normal subjects (N) on a fixed basal diet augmented by a single dose of sodium oxalate (2.6 mmol) given before breakfast

Full size image

Based on the patterns of CaOx supersaturation along the renal tubule, one explanation of these data is that, on the basal diet, the tubular oxalate concentrations at the end of the DLH were likely to be too low in the normals to cause CaOx crystals to “self-nucleate” at that point. Any crystals that might form in normal tubular fluid would only do so towards the end of the CD if CaOx supersaturation in the tubular fluid reached the critical level for “self-nucleation” FP of CaOx. However, any crystals so-formed would not have had time to grow before reaching the end of the CD and would, therefore, be small and easily excreted through the Ducts of Bellini. Even after the oxalate load, the CaOx supersaturation levels in the normals would still be unlikely to reach the FP of CaOx at the end of the DLH and would only become high enough to nucleate spontaneously in the CD. Based on the above reasoning, this would only lead to an increased number of small crystals in the urines of the normals, as observed in Fig. 11. In the RSF, however, because of their higher intestinal absorption of oxalate and subsequently higher urinary oxalate concentrations, CaOx nucleation would be likely to be increased both at the end of the DLH and in the CD. Those crystals initiated at the end of the DLH could potentially be delayed by the various physical and hydrodynamic mechanisms described in the model [59] and under the conditions of the increased tubular concentration of oxalate in these patients give rise to the formation of many more large individual crystals and aggregates, such as observed in the RSF urines after the oxalate load. The much higher RSS CaOx values in the CD of the RSF compared with those in the CD of the normals would also result in a higher crystal growth rate of any small crystals formed at that site and would lead to fewer small crystals actually being excreted compared with the numbers formed on the basal diet as shown in Figs. 10 and 11. Whether or not inhibitors of crystallisation play any role under the conditions of high supersaturation predicted by the model in the transit time between the end of the DLH and the ducts of Bellini, is open to question. Clearly, from the evidence in the “Introduction”, they do not affect “self-nucleation” but it is possible that they might influence crystal growth during the passage of crystals through the distal tubule (DT), where supersaturation decreases slightly [59] before it finally increases again in the CD, or it might affect crystal aggregation at any point in the CD section of the nephron, as predicted from the continuous crystalliser studies described below.

Various other polyanionic compounds have been shown to be effective as inhibitors of CaOx crystal aggregation in the continuous crystalliser system for studying the factors affecting the crystallisation of CaOx [35, 45–47]. These include glycosaminoglycans, ribonucleic acid, the non-polymerised form of Tamm–Horsfall glycoprotein [45–48] and pentosan polysulphate [89]. All of these work by increasing the negative zeta potential on the surface of the CaOx crystals produced, thereby leading to crystal–crystal repulsion and a decrease in crystal aggregation [45–47, 89, 90]. This may also explain the inhibition of aggregation observed in the studies involving pyrophosphate and bisphosphonates [86].

In summary, some polyanionic inhibitors may be important in inhibiting the aggregation of CaOx crystals in urine since they act by changing the charge on the crystal surface, a process that is less likely to be affected by high levels of urinary supersaturation.

Inhibitors of crystal binding to tubular cell epithelium

The interaction between crystals and cells became a popular line of research in the 1990s and 2000s [91–96] and still occupies some workers to this day [97]. It was originally hypothesised by Finlayson and Reid that it was unlikely that CaOx crystals could be initiated and grow sufficiently large to become trapped within the lumen of the tubule within the transit time of urine through the nephron [98] and that stone-formation could only occur on crystals already anchored to the renal epithelium for some reason. However, this assertion has been challenged by a number of workers who have further developed the original kidney model of Finlayson and Reid to incorporate various physical and hydrodynamic factors omitted by them in their original model. This has resulted in an alternative conclusion on the possibility of crystals being trapped in the renal tubules to that drawn by the original authors [59, 88]. Their theory was that crystals formed spontaneously in the tubular fluid had to become attached to the epithelium lower down in the nephron by some unspecified mechanism and, once anchored there, could continue to grow and form the nucleus of an actual stone. Many studies followed using procedures involving layers of various types of tubular cell grown by tissue culture which showed that, under certain conditions, these cell lines could bind crystals of CaOx and that some urinary constituents could inhibit this binding process [91–96]. The most potent inhibitors detected so far are various polyanionic molecules, such as glycosaminoglycans, glycoproteins and citrate [99]. The studies on crystal–cell interactions and the factors influencing these are covered in various excellent reviews on this topic by Khan [97, 100] and I will not elaborate on these now. It is not yet clear if the findings from these studies can be developed into a treatment for preventing CaOx stone-formation.

Inhibitors of crystal growth on randall’s plaques

Since the advent of renewed interest in the role of Randall’s Plaques and Randall’s Plugs in the initiation of calcium-containing stones, much has been written on this topic [101, 102]. However, there has been little research on the possible role of inhibitors of crystallisation in preventing the growth of these entities into stones. Presumably, the same arguments will apply as in “Inhibitors of crystallisation of calcium salts”, “Discussion” and “Conclusions” above but it remains to be seen if there are some more specific factors in urine, other than those already identified, which can influence crystal growth and aggregation on these calcific deposits in the renal papillae.

Discussion

This review clearly demonstrates that excessive supersaturation sufficient to cause the “self-nucleation” of CaOx and CaP in urine or “urine-like” solutions seems to dominate this step in the crystallisation of these salts within the normal 3–4 min transit time of tubular fluid between the glomerulus and the ducts of Bellini. It is also clear that the claims that this step can be inhibited by certain constituents of urine are incorrect since all of the test systems used involved incubation times incompatible with the transit time of tubular fluid through the nephron. At best, some of the inhibitors identified might inhibit the further crystal growth and/or aggregation of the nuclei at lower levels of supersaturation. Thus, the “discovery” of many of these so-called “inhibitors of nucleation” appears to have been made based on a false premise and it is perhaps fortuitous that some of them eventually turned out to be inhibitors of later steps in the crystallisation process.

Unquestionably, urine does possess a certain ability to modify the rate of crystal growth and/or aggregation of calcium oxalate and calcium phosphate crystals and may also contain factors that influence the binding of these crystals to renal epithelial cells. Currently, however, it would seem that the crystal growth and aggregation modifying activity of some of these ions and macromolecules is unlikely to be attributable to one single magic factor X but is probably due to the net effect of all the above promoters and inhibitors (and probably others not yet identified). None of the above factors appears to dominate the kinetics of crystal growth, crystal aggregation and binding of crystals to cells, and none has yet been universally accepted as being uniquely different, either quantitatively or qualitatively, between stone-formers and normal subjects. Therefore, the assertion that a deficiency in one particular inhibitor or an excess of one particular promoter is THE cause of stone formation remains open to question. In the final analysis, stone-formation is probably due to the abnormal combination of factors that influence both the thermodynamic driving force (supersaturation) and the kinetic (rate-controlling) processes involved in the crystal growth and aggregation of the various stone-forming minerals. For some types of stone-formation, such as cystine, xanthine, 2,8-dihydroxyadenine, uric acid and magnesium ammonium phosphate, the thermodynamic factors appear to dominate the process; for calcium-containing stones both sets of factors may be involved but the effectiveness of the inhibitors on crystal growth appear to be largely dependent on the level of supersaturation of the urine with respect to the calcium salt concerned. However, inhibitors may play a role in influencing the degree of aggregation of the crystals produced which is independent of the level of supersaturation.

Conclusions

The evidence examined in this critique would suggest the following:

  1. 1.

    So-called inhibitors of “self-nucleation” do not play any role in preventing the initiation of the crystallisation of CaOx or CaP in urine because excessive urinary supersaturation can overwhelm their effect on the “self-nucleation” of these salts within the normal time frame of the transit of urine through the nephron (3–4 min).

  2. 2.

    Inhibitors of the crystal growth of CaOx crystals may or may not play a significant role in the prevention of CaOx stone-formation since once again excessive urinary supersaturation can overwhelm the effect of the inhibitors on the growth process. However, they may play a role as inhibitors of crystal growth at lower levels of metastable supersaturation where the balance between supersaturation and inhibitors as competitive driving forces in the crystal growth process is more even.

  3. 3.

    Inhibitors of CaOx crystal aggregation may play a significant role in the prevention of stones, since they do not seem to be strongly affected by excessive supersaturation, at least in vitro. There is also some evidence that at least some of these inhibitors may not be affected by excessive supersaturation in vivo.

  4. 4.

    Inhibitors of CaOx crystal binding to renal tubular epithelium may exist but further studies are necessary to elucidate their importance in the actual formation of stones in the urinary tract.

  5. 5.

    There may be an alternative explanation than a deficiency in the excretion of inhibitors for the observations in those studies which have shown a difference in the size and degree of aggregation of CaOx crystals in the fresh, warm urines collected from normal subjects and patients with recurrent CaOx stones. This may depend more on the site of “self-nucleation” of CaOx crystals in the renal tubule rather than to differences in the excretion of so-called inhibitors of crystallisation between the two groups.

  6. 6.

    The claims that administration of potassium citrate, potassium magnesium citrate or magnesium hydroxide reduces the rate of stone recurrence may be more due to the effect of these forms of medication on the supersaturation of urine with respect to CaOx and CaP than to any increase in “inhibitory activity” attributed to these treatments.

  7. 7.

    In summary, there is a competition between supersaturation and so-called inhibitors of crystallisation which ultimately determines the pattern of crystalluria in stone-formers and normals. If the supersaturation of urine with respect to CaOx reaches or exceeds the 3–4 min formation product of that salt, then it dominates the crystallisation process both in terms of “self-nucleation” and crystal growth but may have little or no effect on the degree of aggregation of the crystals produced. As the level of supersaturation of urine with respect to CaOx decreases within the metastable region, the influence of inhibitors increases and some may affect both the crystal growth and aggregation of any existing crystals of CaOx.