Inhibitors and Promoters of Stone Formation

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Kidney International, Vol. 13 (1978), pp. 361—3 71

Inhibitors and promoters of stone formation

HERBERT FLEISCH

Department of Pathophysiology, University of Berne, Berne, Switzerland

Currently, three main mechanisms are thought to Crystal growth and crystal aggregation be important in the formation of urinary stones: 1) In the past, attention was devoted mostly to the the relationship between the concentration of the formation and growth of crystals. Recently, interest precipitating substances in urine and the solubility of has been directed to an area which, until now, had the mineral phase formed, 2) the role of promoters of been neglected: the crystal aggregation. This term crystallization and aggregation, and 3) the part describes the process of crystals binding one to an- played by inhibitors of crystal formation and aggre- other, resulting in the formation of larger clusters. In gation (Fig. 1). vitro, aggregation of both calcium oxalate [16, 17] Saturation of urine and calcium phosphate crystals [18] occurs readily when the solution is supersaturated. Aggregation It is now widely accepted that even in normal could be the mechanism which distinguishes simple people, urine is ordinarily supersaturated with re- crystalluria, which occurs in most normal people, spect to calcium oxalate [1—6], octocalcium phos- from stone formation. This line of thought is phate [2, 3], hydroxyapatite [1, 2], and sometimes strengthened by the finding that while usually only with respect to brushite [1, 7, 8]. The degree of individual calcium oxalate crystals are found in nor- supersaturation is usually higher in patients with mal people, stone-formers often excrete large aggre- urinary stones [2—4, 7—10]. This is due mainly be- gates of this salt [16, 19]. cause these patients tend to excrete more calcium [10—12], but also because urinary oxalate can be Promoters of crystallization increased [10, 13]. Urine is also often supersaturated A few years ago, great emphasis was placed on the with respect to sodium urate and ammonium urate supposed activating role of organic compounds in [14, 15]. Supersaturation with regard to magnesium urine [20]. This was based on the finding that urinary ammonium phosphate is restricted to the cases proteins bind calcium [21] and can induce calcifica- where the urine becomes alkaline because of ammo- tion in vitro [22] under certain conditions. Further- nium production by bacteria [2] (GRIFFITH, this more, proteins are increased in amount [22, 23] and issue). are qualitatively different [24, 25] in urine samples Supersaturation can vary in degree. It can be in the from stone-formers than in that from normal people. metastable range where precipitation may occur only Such an activating theory was invoked to account for when induced by epitaxy or heterogenous nuclea- the close morphological relation existing between the tion, or it can be in the unstable region where rapid matrix and the mineral in the stones [26, 27]. All spontaneous precipitation does occur. The limit be- these results, however, give no evidence that the tween the two ranges, which can be called the spon- change in the urinary proteins is the primary event in taneous formation product, is not a fixed number but stone formation. Indeed, they just as well might be will depend upon the duration of incubation. secondary to the irritation from the stone and to These theoretical considerations are relevant to infection. The morphological relation between ma- what occurs in vivo. It was found that when the trix and mineral could be due to a co-precipitation of urinary saturation measured chemically is above the the proteins with the mineral or their secondary spontaneous formation product required for a rapid binding onto the crystals [28]. Lately, the theory of induction of precipitation in inorganic solutions, crystals can usually be detected in the voided urine specimen [3]. On the other hand, when the ion prod- 0085—2538/78/0013—0361 $02.20 uct is below this product, crystals are absent. © 1978, by the International Society of Nephrology.

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cretion of urate, reduces the formation of calcium I SUPERSATURATED SOLUTION stones [34, 35] (seeCOE,this issue). 4 Homogenous/heterogenous nucleation Inhibitors of crystallization and crystal growth Since urine usually is supersaturated with respect to the various stone-forming salts and contains crys- SMALL CRYSTALS tals, the conditions for crystal formation, aggrega- 9'- tion, and thus stone formation are satisfied. Thus, Crystal growth Crystal aggregation the main question is not why stones can form but 4 1 why stones do not form more generally. One explanation might be the presence in urine of very effec- I LARGECRYSTALS -.0.1 CRYSTALAGGREGATES I tive inhibitors for both these processes, crystal for- t mation and aggregation. Fig.1. Mechanisms of mineral formation. Inhibition of crystal growthis denoted by open arrows; inhibition of crystal aggrega- Inhibitors of ihe formation of calcium phosphate tion is denoted by the large closed arrows. Chemical estimation of inhibitory activity. One of the first techniques testing urine's inhibitory capacity the activating role of urinary proteins has been aban- [36, 37] made use of a system, described in the doned by most investigators. As such a role, how- 1930's, for studying the calcification of cartilage [38]. ever, could not be proven, it also has not been Epiphyseal cartilage from rachitic rats was incubated disproven. in vitro in a supersaturated salt solution, and the In recent years, emphasis has been shifted towards precipitation of calcium phosphate was studied. Ur- the role of crystals of one salt inducing the crystalli- ine or the substances to be tested are added to the zation of a salt of another nature. Such an epitactic incubation fluid. The results, however, are difficult induction between crystals having similarities in lat- to interpret. Since the cartilage is enzymatically very tice dimensions is a well known phenomenon in active, it is likely to destroy certain inhibitors. Fur- crystallography. Relevant lattice similarities are thermore, the test substance or urine may influence present between uric acid, calcium oxalate, and cal- the enzyme activity as well as the metabolic ma- cium phosphate crystals [29], and epitactic induction chinery of the incubating cartilage, thereby altering does occur among them. Thus, the precipitation of the precipitation by a mechanism unrelated to crystal sodium urate is favored by both hydroxyapatite and growth inhibitors [39]. Obviously, the results ob- calcium oxalate [30]. The precipitation of calcium tained can be totally unrelated to stone formation, oxalate can be induced from a metastable solution by and the technique, while possibly useful for cartilage hydroxyapatite [30, 31], brushite [30], and urate [30, calcification, should be abandoned for the study of 32, 33] but not consistently by uric acid [30, 33] (see stone formation. Dr.Coe's article, this issue). Conversely, calcium A theoretically correct approach of practical im- phosphate precipitation is stimulated by both cal- portance is the determination in vitro of the mini- cium oxalate and monosodium urate crystals [30, mum product of calcium x phosphate (formation 33], while uric acid is not effective [30, 33]. Interest- product) necessary for the formation of a solid phase ingly, this effect of calcium oxalate on calcium phos- within a preset time period under defined conditions. phate precipitation is not as efficient as the reverse The experimental conditions of this technique, which [31]. This might be because hydroxyapatite is not the measures mainly heterogenous nucleation (see FIN- first salt to form when calcium phosphate precipi- LAY5ON, this issue), are critical and need to be care- tates, but is preceded by other phases, the nature of fully controlled. For example, the time of incubation which is still disputed. These initially formed phases has an inverse effect: the shorter the time of incuba- spontaneously transform into the more stable tion the greater the formation product [40]. Inhibi- hydroxyapatite. tors will increase this product. It has been reported This epitactic mechanism of precipitation could that inhibitors are not active at very short incubation give an explanation for the well known fact that most times, and that they increase the formation product stones are formed not just by one salt but by a value only up to that obtained at short incubation mixture of different kinds of salts. It could also times [40]. It has been suggested that this occurs explain the clinical findings that patients with cal- because homogenous nucleation is predominant ini- cium stones are often hyperuricosuric [34] and that tially, and is later replaced by heterogenous nuclea- treatment with allopurinol, which decreases the ex- tion. However, as discussed in Finlayson's article Inhibitors and promoters of stones 363

(this issue), it is unlikely that homogenous nucleation measurement of inhibitory activity in diluted urine could proceed in complex solution such as urine. may not be representative of whole urine. Since Moreover, the report showing the independence of inhibitory activity is generally not related linearly formation product from inhibitor activity has not with its concentration, and since the concentration- been confirmed [411. effect relation is different for the various inhibitors, It must be kept in mind that the determination of their relative importance will change with dilution. the formation product does not measure specifically Furthermore, urine itself can influence the effect of an effect on the formation of the final phase, since in certain inhibitors so that an effect can be different in some cases there is first one salt formed which then inorganic solutions, diluted urine, and whole urine. transforms into a salt of another type [421. Thus, an Another approach which measures mainly crystal inhibitor acting on the growth of the second salt growth is to measure the kinetics of the precipitation might be missed since the first will be formed nor- of the mineral after addition of a seed to trigger the mally. The nature of the first phase is still disputed. reaction. Calcium phosphate [55] or other nucleating Amorphous calcium phosphate [43, 44], brushite materials are added to a solution with a defined [45—471, octocalcium phosphate [40, 45, 48, 49] are supersaturation, and the decrease in the solution of some of the proposed forms. calcium or phosphate concentration or both are mea- The formation product can also be determined in sured. At acid pH, where brushite is formed, the rate the presence of a nucleating agent of some kind, such of crystal growth is proportional to the square of the as collagen 150], elastin, or crystals of another salt. supersaturation, allowing one to calculate readily the Nucleating agents will decrease the formation prod- rate of crystal formation [56]. Unfortunately, at uct for a given incubation time and permit the detec- higher pH, where salts other than brushite are tion of inhibitory activity at shorter incubation times. formed, the precipitation curve obtained is more While very useful to investigate the effect of indi- complex and cannot be analyzed quantitatively, vidual compounds, the determination of the forma- probably because of the formation of precursor tion product presents problems when used to test phases which later transform into more stable ones, urine. In urine, one must measure the thermody- especially apatite [42, 55]. Unfortunately, it is these namic product of the ion activities and not just the conditions which are likely to be more relevant phys- simple calcium x phosphate product. Various means iologically, since although brushite is thought by have been investigated to determine this product. some to be the first salt to form in calcium phosphate One is to determine chemically all the ions and com- stones [47, 57], it is normally not found in the formed plexes involved and to calculate their activities in the stones. A simplified version of this approach is to urine using the chemical stability constants described determine just the amount of calcium salt formed in the literature [2]. This procedure is tedious and within one chosen time [9, 46, 57—60]. probably not without error, since some of the stabil- In both these approaches it is important that the ity constants are still doubtful [51]. A simplified supersaturation level is well defined. As discussed by approach has been suggested which consists of mea- Finlayson (this issue), in whole urine, the compari- suring the formation product as the ionic concentra- son of the ratio of the Ca x P1 versus the Ca x P, tion of calcium times phosphate (Ca2 x HPO, or measured after incubating with brushite, is probably Ca >

Methylene blue had no effect [84]. Diphosphonates support for such a surface controlled mechanism is are active inhibitors also when given in vivo [105]. the fact that inhibition of the formation rate of brush- The effect of pyrophosphate and the diphosphonates ite [63] and calcium oxalate [93] follows the laws of increase at alkaline pH, while the effect of urine is the Langmuir adsorption isotherms. Inhibition of ag- not pH-dependent [85]. gregation, on the other hand, is more likely to be due It has been difficult up to now to assess the amount to a change in the zeta potential of the surface, which of the total inhibition played by the various parts. will alter attraction or repulsion between crystals. Some data indicates that pyrophosphate would account for about 15% of the total activity [1061. A large part would be due to a macromolecule of large Inhibitors in patients with urinary stones molecular weight [85, 107] which is precipitated by The question as to whether stone formers have cetyl pyridinium chloride, which suggests that it less inhibitors in their urine is still not completely could be a glycosaminoglycan [106]. settled, although there is now strong evidence that many patients do show such a defect. This uncer- tainty may be attributed partly to the wide variety of Mechanism of the inhibitory activity techniques used, insufficient data available from Most of the work on the mode of action of the study of whole urine, and the failure often times to relevant inhibitors has been done on pyrophosphate obtain control groups, matched with respect to age, and diphosphonates. These compounds were found sex, and diet. to delay the various processes involved in the forma- A decrease of inhibitory activity of calcium phos- tion of the solid phase, namely epitactic or heteroge- phate precipitation has been described in stone-for- nous nucleation, crystal growth, crystal aggregation, mers, using the rat cartilage system [36, 37, 68, 74]. and phase transformation from the amorphous to the A decrease has also been found using the determina- crystalline form. These various effects appear to be tion of the Ca x P1 formation product, either in related to the binding of the inhibitors onto the crys- presence of diluted urine [1341 or in whole urine [91. tal surface where they inhibit induction of new crys- For calcium oxalate, a decrease in inhibitors was tals, growth, and aggregation. The relation between detected, using the determination of the formation the absorption of substances onto the crystals and product [91, as well as the crystallization [5, 9, 94] in their effect on crystal growth is a well known phe- whole urine. A decrease in inhibition of calcium nomenon. Many compounds, called crystal modi- oxalate aggregation has been described in urine of fiers, can inhibit the crystal growth of certain salts, stone patients [4, 16, 114]. This is especially the case and there is a close correlation between the concen- in patients presenting a relatively smaller urinary tration of the compounds on the crystal surface and calcium x oxalate supersaturation, so that when the degree of inhibition [108]. It is not necessary, both parameters, supersaturation and inhibition of however, for the whole surface to be covered by a aggregation, are taken into account stone-formers macromolecular layer in order to obtain an inhibi- and controls can be very clearly separated [1141. tion. Specific binding onto the sites of crystal growth Various data indicates that pyrophosphate is de- will suffice, so that an inhibition is obtained when creased in stone-formers [73, 115, 116], although less than one percent of the surface is covered. such a decrease has not been found by all investiga- Pyrophosphate and diphosphonates have both tors [691. The observed difference seems to occur been shown to be bound strongly onto the crystals of mainly in middle-aged men [73], a group in which hydroxyapatite [109—111]. The amount bound can be stone formation is actually most frequent, and in the very large (for pyrophosphate, about two molecules patients without hypercalciuria [116]. The lack of per surface unit cell [109, 111]), and is accompanied pyrophosphate becomes more apparent when pa- by a displacement of orthophosphate [111—113]. tients are matched to control with respect to age, From the results obtained by Scatchard plots, it sex, and diet, in which condition, a diminution of appears that the binding is not restricted to a single 50% has been observed in men [134]. Women, how- class of binding sites with constant affinity [111]. ever, showed no abnormality. Interestingly, the affinity to the first site goes in Until now, only a few ways to increase urinary parallel with the inhibitory activity of crystal forma- inhibitors are known. As will be discussed in Dr. tion [1111, suggesting it represents binding onto the Thomas's article (this issue) one of them is the oral sites responsible for crystal growth. It is likely that administration of orthophosphate, which will in- the binding occurs through a calcium ion. A further crease the excretion of pyrophosphate [117, 118]. Inhibitors and promoters of stones 367

Cellulose phosphate, however, does not [52]. Ortho- ment of a phosphonate which is excreted in urine but phosphate administration decreases both calcium ox- does not enter bone might solve this problem. alate and calcium phosphate aggregation in urine In the studies of stone formation, it is necessary, [119]. Furthermore, it is claimed by many [120— however, to keep in mind that urolithiasis is most 124], although not accepted by all [125, 1261, to be probably a multifactorial disease. Several predispos- efficient in the treatment of recurrent calcium stones. ing factors, such as hypercalciuria, hyperoxaluria, All these effects could be explained at least partly by hyperuricosuria, urinary infection, deficiency of in- the increase in urinary pyrophosphate, although the hibitors of crystal formation, and deficiency in inhibi- decrease in calciuria which occurs and which de- tors of crystal aggregation can play a role. Not all are creases in turn the urinary saturation vs. calcium likely to be present in the same patient, so that it will oxalate [1191 is also likely to play a role. The satura- become necessary to divide the patients into tion vs. calcium phosphate, however, is not altered subgroups, according to their pathophysiological dis- [119] nor is it increased [52]. Recently, thiazide, turbance. The same is likely to be true for the treat- which has been proposed in the treatment of stones ments. A specific treatment for one disturbance can because it decreases urinary calcium [34, 127], has be beneficial in this group, but possibly not in others. been shown to increase the formation product in This basic principle is frequently disregarded which whole urine both of calcium oxalate [128] and brush- explains why the treatment is most often far from ite [129]. This effect is probably due to the increase satisfactory. of pyrophosphate following the increase in ortho- Summary phosphate excretion. As will be discussed in the The understanding of the formation of urinary article by Yendt and Cohanim (this issue), renal stones centers around three main mechanisms: the excretion of other inhibitors, such as zinc, may be urinary concentration of stone-forming ions, the role increased as well. of promoters, and the role of inhibitors of crystal The parallelism between the excretion of ortho- formation and crystal aggregation. With respect to phosphate and pyrophosphate is probably due to a the promoting activity, lately emphasis has shifted direct action of orthophosphate on the kidney. In- from the role of the organic matrix to that of one salt deed, the oral administration of orthophosphate inducing by epitaxy the precipitation of another salt. causes a rapid increase in the renal clearance of Among the inhibitors, it has become necessary to pyrophosphate without a change in the blood con- distinguish between those affecting crystal formation centration [130]. Furthermore, as shown in studies and those affecting crystal aggregation. For measur- with dogs, the unilateral infusion of orthophosphate ing the inhibitory activity, the various techniques and into one kidney causes a greater and more rapid their relevance have been reviewed. It has been increase in the infused kidney than in the noninfused found that the main inhibitors for calcium phosphate one [1301. and calcium oxalate precipitation are citrate, pyro- The powerful effects of the diphosphonates on the phosphate, and perhaps magnesium. Those for cal- formation and aggregation of calcium phosphate and cium phosphate and calcium oxalate aggregation are calcium oxalate crystals and the fact that they are glycosaminoglycans, pyrophosphate, and citrate. excreted in the urine after oral administration raised Among the synthetic inhibitors, the diphosphonates the possibility that these compounds might be useful are the most powerful for both processes. The role in the treatment of urinary stones. In animal studies, and the therapeutic implications of these various they were found when given orally to decrease the concepts have been discussed. formation of experimentally induced bladder stones [104]. In man, the oral administration of ethane-l- Acknowledgment hydroxy-1,1-diphosphonate (EHDP) decreases the This work was supported by the Swiss National amount of calcium oxalate crystal aggregates [1051, Science Foundation (3.725.76) and by the Procter increases the formation product of calcium phos- and Gamble Company, U.S.A. phate when tested in diluted urine [131], and decreases the growth of brushite in full urine [132]. Reprint requests to Prof. H. Fleisch, Department of Pat hophys- Clinically, it appeared to decrease the recurrence of iology, University of Berne, Murtenstrasse 35, 3010 Berne, stones [131]. However, the dosage needed to be Switzerland. effective was at a level where an effect on bone References turnover and mineralization was likely, so that its 1. YARBROCL:Studies on the mechanism of formation of renal use does not seem to be warranted. The develop- calculi: Part II. J Urol 80:10—12, 1958 368 Fleisch

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