Initiation and Growth of Uroliths

Canine Urolithiasis I

Initiation and Growth of Uroliths

David F. Senior, B.V.Sc.,* and Birdwell Finlayson, M.D., Ph.D. t

Three factors predispose to urolithiasis: (1) a high degree of urinary supersaturation; (2) reduced urinary inhibition of crystal growth; and (3) prolonged particle transit time in urine. 6 This article will address all three factors after briefly examining the composition of uroliths. COMPOSITION OF UROLITHS

Uroliths are polycrystalline concretions composed primarily of organic or inorganic crystalloids and smaller quantities of organic matrix. 2 • 12• 34 Cross sections of uroliths usually reveal nuclei and laminations. On occasion, radial striations may be apparent. A variety of different minerals may be present in the uroliths of dogs (Table 1). 1· 4• 5 · 8 • 10· 14 • 32• 33 Struvite is observed most frequently, whereas the relative incidence of calcium oxalate, cystine, and ammonium urate tends to vary between surveys. Only two surveys were based on accurate crystallographic or x-ray diffraction methods. 1· 14 In most uroliths, only one mineral type is represented, but on occasion, different minerals may be mixed or one mineral may be present inside, with outer layers composed of a second mineral. When a urolith is demineralized by immersion in a mild solvent, organic matrix remains. From the center to the periphery of the stone, there appears to be progressively more matrix, then it becomes less dense, which gives the cut section a laminar appearance. 25 Electron micrographic examination reveals a meshwork of proteinaceous material with both amorphous and fibrous characteristics. 16 In humans, the basic composition of matrix is similar for all stones regardless of crystalline composition. 25 Organic matrix substances identified in human uroliths and uroliths experimentally produced in animals include matrix substance A, Tamm-Horsfall mucoprotein, uromucoid, serum albumin, alpha and gamma globulins, and chon*Associate Professor, Department of Medical Sciences, University of Florida College of Veterinary Medicine, Gainesville, Florida tProfessor, Department of Surgery, University of Florida College of Medicine, Gainesville, Florida Veterinary Clinics of North America: Small Animal Practice-Vol. 16, No. 1, January 1986

19

20

DAVID F . SENIOR AND BIRDWELL FINLAYSON

Table 1. Incidence of Different Types of Uroliths in the Dog DATE PUBTOTAL LISHED EXAMINED

AUTHOR

STRUVITE

URATE

CYSTINE

OXALATE

OTHER

Brodey 4 (United States)

1955

52

42

10

White33 (United Kingdon)

1966

350

211

18

67

54

Finco 10 (United States)

1970

73

62

6

5

0

Weaver32 (United Kingdon)

1970

100

53

13

20

14

Clark" (United Kingdom)

1974

110

49

2

24

35

Brown5 (United States)

1977

438

307

21

95

12

3

Hicking 14 (West Germany)

1981

299

151

16

37

4

18*

Bovee 1 (United States)

1984

272

187

19

9

27

30*

TOTAL

1694

1062 (63%) 105 (6%) 257 (15%) 146 (9%) 51 (3%)

*These surveys used accurate crystallographic or x-ray diffraction methods of stone analysis.

droitin sulfuric acid. 34 Matrix substance A comprises the bulk (84 to 88 per cent) of the total organic matrix. 25 Substance A is not a single entity but rather is made up of three or four different proteins. 18 Interestingly, substance A is similar to a calcium-binding protein obtained from the intestinal mucosa of the chick. 31 INITIATION AND GROWTH OF UROLITHS The formation of uroliths involves crystal nucleation and growth in a solution. Understanding of the physical chemistry of solutions enhances understanding of the mechanisms involved in urolith formation . The concentration of ions in solutions is easily measured by standard chemical techniques. However, the chemically measured value is not a true indication of the effective cbncentration, which is called the "activity." In a complicated solution like urine, an individual ion may complex with many other ions according to known dissociation constants. For example, magnesium complexes with phosphate, citrate, and oxalate. Therefore, the free-ion concentration of magnesium can only be estimated if all dissociation constants for all significant complexes are known. Also, free or noncomplexed ions are further restricted in their motion by the nonspecific effect of other ions in solution, an effect represented by the term "activity coefficient." The activity coefficient varies with the electrical field strength

21

INITIATION AND GROWTH OF UROLITHS

of a solution. In the usual concentrations found in urine, the activity coefficient for monovalent ions is about 0. 7; for divalent ions, it is about 0.3. 26 Computer programs that estimate ion activity from chemical concentration measurements and take into account the formation of inter-ion complexes and activity coefficients are available. 13 • 20 • 28 The product of the activities of ions in a precipitating salt solution is called the activity product. As the activity product of electrolytes is increased in aqueous solution, a number of well-defined regions of precipitation can be identified (Fig. 1). 19 When a solution in contact with precipitated crystals is saturated, the solid phase is in equilibrium with the liquid phase. The rate of crystal growth equals the rate of dissolution, and the crystal mass remains the same. At this point, the activity product of the ions in solution is called the "thermodynamic equilibrium solubility product" (K.P). If the activity product is reduced below the K.P' the rate of dissolution will exceed the rate of growth and the crystal will dissolve. If the activity product is raised above the K.P' the crystal will grow. The rate of growth or dissolution is proportional to the degree to which the activity product exceeds or is lower than the K•P' respectively. K.P varies with temperature. This phenomenon provides an explanation of why crystals in urine tend to grow in size and number if the specimen is refrigerated. When the activity product of a solution is raised above the Ksp in absence of crystalline material, a region of metastable supersaturation is reached wherein crystal growth will only take place upon the addition of crystalline material or another surface that can act as a template for crystal growth (see Fig. 1). Spontaneous crystal formation will not occur. If the activity product is increased further, a poorly defined level of critical supersaturation is reached above which spontaneous crystal precipitation can occur in the labile region. The activity product at critical supersaturation is known as the formation product. Canine urine is frequently supersaturated with respect to a number of crystals, including struvite, hydroxylapatite, ammonium urate, and calcium

LABILE REGION Figure 1. The effect of increasing activity product on solution saturation. (From Nancollas, G. H .: The kinetics of crystal growth and renal stone formation . In Fleish, H., et al. (eds.): Urolithiasis Research. New York, Plenum Press, 1976; with permission.)

t

CRITICAL SUPERSATURATION

INCREASING

METASTABLE SUPERSATURATION

PRODUCT

SATURATION, K 5 P UNDERSATURATION

ACTIVITY

22

DAVID F . SENIOR AND BIRDWELL FINLAYSON

oxalate. These crystals are often observed on routine urinalysis in normal dogs. It is not known if canine urine is often above the formation product for these minerals. NUCLEATION The initial process of urolith formation consists of precipitation of small submicroscopic aggregates of ion groups in a process called nucleation. Nucleation corresponds to the production of new centers from which growth can occur. The driving force for nucleation is supersaturation, whereas the restraining force is the energy requirement for forming new surface. 19 As nucleation creates a solid-liquid surface or interface, surface tension tends to tear small groups of particles apart. 19 The magnitude of the effect of surface tension is inversely proportional to the radius, so that larger groups of particles have less of a tendency to disperse back into solution. 19 Once a critical size nucleus is formed, its composite groups of particles remain stable and can continue to grow (Fig. 2). The probability that groups of particles that are larger than critical size will form increases at greater degrees of supersaturation 19-that is, when the driving force exceeds the restraining force, nucleation occurs. Solutions in the labile range of supersaturation readily generate critical size particles and thus permit spontaneous nucleation. 19 Because nucleation occurs in this setting without involvement of pre-existing particles or surfaces, this process is known as homogeneous nucleation. 19 Heterogeneous nucleation occurs when crystal nuclei form on a pre-existing surface. 19 This process can take place in

0

+

0

-

co

+

0

-

ce

-

ffi

-

on

ce

+

0 I

I

on_,+ 0

critical nucleus

Figure 2. Diagrammatic illustration of the formation of a critical size nucleus by homogeneous nucleation. (From Nancollas, G. H. : The kinetics of crystal growth and renal stone formation . In Fleish, H., et a!. (eds.): Urolithiasis Research. New York, Plenum Press, 1976; with permission.)

INITIATION AND GROWTH OF UROLITHS

23

metastable supersaturation. The new crystal nucleus only has to overcome part of the interfacial surface tension to form, so nucleation can occur at a lower degree of supersaturation.9 Because precipitation is more likely if heterogeneous nucleation occurs in urine, many attempts have been made to identify possible nucleation surfaces or niduses in the urine of urolith-formers. Foreign bodies such as suture material and indwelling catheters are well-established nucleation surfaces. 30 However, in the absence of such obvious initial surfaces, niduses have been difficult to identify. Nucleation may occur on another crystal type in a process known as epitaxy. 17 Here, a complementary match between the crystal lattices of two minerals has been looked for, but there is infrequent evidence of the occurrence of this phenomenon in urine. Matrix has been implicated as the initial surface upon which heterogeneous nucleation may occur, but the role of matrix remains unclear. 34 Necrotic debris and desquamated cells have been suggested, but they have not been found at the central stone nucleus. 25 In summary, nucleation may occur spontaneously or on a pre-existing nidus. It is emphasized that the nature of niduses is presently unclear. Irrespective of the mechanism of nucleation, precipitation is more likely if urine is concentrated. Extremes of urinary supersaturation occur for (l) struvite in dogs with urease-producing bacterial infection, (2) cystine in dogs with congenital cystinuria, and (3) ammonium urate in dogs with hepatic and renal urate transport defects. 15• 23 Urolithiasis is common in all three instances. CRYSTAL GROWTH

Once nucleation has occurred, crystal growth may continue provided the surrounding fluid remains supersaturated. Stone particles grow by the addition of further ion groups onto the surface or by aggregation of crystals.9 At low supersaturation, the addition of further atoms or ion groups is thought to take place at spiral fault lines on the crystal surface. 9 Deposition at a fault line requires less energy than commencement of a new layer. At high supersaturation, addition of further atoms or ion groups is more random and may occur anywhere on the surface of the crystal. Aggregation or coalescence of formed crystals can cause an extremely rapid increase in the size of particles. Small particles tend to aggregate readily because in the aggregation process, the surface-to-mass ratio is reduced, causing energy to be released.9 Because small particles in solution frequently collide, there is ample opportunity for aggregation. Inhibitors of the growth and aggregation of calcium phosphate and calcium oxalate crystals have been identified in human urine. 6 Citrate, pyrophosphate, chondroitin sulfate, acidic peptides, and ribonucleic acid have inhibitory activity, although the relative importance of each has not been clearly established. 3 Inhibitors appear to act by occupying growth sites on the surface of crystals, thereby preventing the addition of further atoms or ion groups. 9 • 19 Inhibitors exert this effect at very low concentration. The same inhibitors of the growth of calcium phosphate and calcium oxalate crystals in humans are probably present and active in canine urine.

24

DAVID F. SENIOR AND BIRDWELL FINLAYSON

Inhibitors of crystal growth appear to be important in stone disease in humans. Human calcium oxalate urolith-formers and non-stone-formers cannot be distinguished on the basis of urinary supersaturation alone. However, when urine inhibitory activity and supersaturation are taken into account, there appears to be a clear-cut division between the two groups 21-that is, both groups have individuals with relatively high and low supersaturation, but high saturation combined with low inhibitory activity in urine clearly identifies persons at risk for formation of uroliths. Conversely, low supersaturation with high inhibitory activity identifies people with a low risk of urolith formation. Thus, the balance between the degree of supersaturation and urinary stone growth inhibitory activity determines the likelihood of calcium oxalate urolithiasis in man. The concentration of inhibitors in urine may influence formation of ammonium urate uroliths in the Dalmatian dog. Normally, Dalmatians form urine that is well in excess of the formation product for ammonium urate. 23 Spontaneous nucleation occurs, but a fine colloidal suspension, rather than coarse precipitation, results. This is thought to occur because the small nuclei are extremely hydrophobic. The ammonium uratecolloidal particles move harmlessly through the urinary tract with surrounding urine. 24 Fiocculation or coalescence of the colloidal particles may occur if the activity of urate or, more importantly, NH 4 + is increased beyond a certain level. 24 Macromolecular inhibitors of flocculation of ammonium urate colloids have been identified in urine of Dalmatians.22 Inhibition of aggregation may be mediated by coating the sites of attachment between colloidal particles or by altering the surface charge on the particles so they repel one another. Inhibitors of the growth of struvite and cystine crystals have not been identified. TRANSIT TIME Urine supersaturation and crystalluria are normal phenomena in dogs. As long as crystals continue to move at the same velocity as urine, stone disease does not occur. From the point of view of physical chemistry, it is unlikely that a crystal particle could grow to a size large enough to delay its passage down the ureter unless some factor delayed its passage, allowing growth to continue. 11 Thus, nephroliths are most likely the result of fixed particles that initially grow in situ and then break off into the renal pelvis. Randall's plaques are accumulations of calcium crystals in subepithelial portions of medullary collecting tubules. These have been incriminated as a starting point for formation of calcium oxalate uroliths in human kidneys. 11 Crystals in urine do not readily adhere to the uroepithelium. This phenomenon is thought to be due, at least in part, to glycosaminoglycans that coat the surface of the uroepthelium. 7 Disruption of the uroepithelial surface allows crystal adherence. 7 Perhaps struvite nephroliths that occur secondary to urinary tract infection in dogs become established because ammonia produced by bacteria alters the uroepithelial surface, promoting crystal adherence. 21 On the other hand, urolith formation in the urinary bladder seems feasible without invoking the need for a fixed-particle mechanism. 11 The

JNJTIATION AND GROWTH OF UROLITHS

25

pet that is house-trained well and left for long periods between urination may be predisposed to prolonged crystal transit time and poor emptying of the bladder. Both situations may represent risk factors for urocystolithiasis. Animals with an atonic bladder usually have a large residual bladder volume after voiding. Larger crystals may settle in the ventrum of the bladder and continue to grow if not voided. Eventually, a stone may grow to a size that will not readily pass through the urethra. In summary, the ability to form hyperosmolar (concentrated) urine is a common occurrence in terrestrial animals. Unfortunately, some of the solutes in urine are sparingly soluble. When water is removed from tubular fluid that traverses the collecting tubules, an environment conducive to stone disease develops. Supersaturated urine becomes a driving force that leads to crystal nucleation and subsequent growth of crystals. Extremes of urinary supersaturation occur in dogs with urease-producing infection, cystinuria, and defects in urate transport. Nucleation occurs more readily if surfaces are available for deposition. The rate of crystal growth is dependent on the degree of supersaturation. Crystal growth may be inhibited by natural substances in urine. The balance between supersaturation and inhibition of crystal growth determines whether or not uroliths will form, at least in a calcium oxalate system. If crystals are delayed in their transit through the urinary tract, they may grow to such a size that they cannot readily pass through the ureters or urethra.

REFERENCES 1. Bovee, K. C. , and McGuire, T. : Qualitative and quantitative analysis of uroliths in dogs: Definitive determination of chemical type. J. Am . Vet. Med. Assoc., 185:983-987, 1984. 2. Boyce, W. H., and Garvey, F. K. : The amount and nature of the organic matrix in urinary calculi. A review. J. Urol., 76:213-227, 1956. 3. Breslau, N. A. , and Pak, C. Y. C .: Urinary saturation, heterogeneous nucleation, and crystallization inhibitors in nephrolithiasis. In Coe, F . L. (ed.): Nephrolithiasis. New York, Churchill Livingstone, 1980, pp. 13-36. 4. Brodey, R. S.: Canine urolithiasis. J. Am . Vet. Med. Assoc., 126:1-9, 1955. 5. Brown, N. 0., Parks, J. L., and Green, R. W. : Canine urolithiasis: Retrospective analysis of 438 cases. J. Am. Vet. Med. Assoc., 170:414-418, 1977. 6. Burns, J. R., and Finlayson, B.: Why some people have stone disease and others do not. In Roth, R. A., and Finlayson, B. (eds.): Stones. Clinical Management of Urolithiasis. Baltimore, Williams & Wilkins Co., 1983, pp. 3-7. 7. Chang, S. Y., Gill, W. B., and Vermeulen, C. W.: Povidine-iodine bladder injury in rats and protection with heparin. J. Urol. , 130:382-385, 1983. 8. Clark, W. T. : The distribution of canine urinary calculi and their recurrence following treatment. J. Small Anim. Pract., 15:437-444, 1974. 9. Coe, F. L.: Physical chemistry of stone formation. In Coe, F. L. (ed.): Nephrolithiasis. Pathogenesis and Treatment. Chicago, Year Book Medical Publishers, Inc., 1978, pp. 27-47. 10. Finco, D . R., Rosen, E ., and Johnson, K. H.: Canine urolithiasis: A review of 133 clinical and 23 necropsy cases. J. Am. Vet. Med. Assoc., 157:1225-1228, 1970. 11. Finlayson, B.: Where and how does urinary stone disease start? An essay on the expectation of free- and fixed-particle urinary stone disease. In Van Reen, R. (ed.): Idiopathic Urinary Stone Disease. Fogarty International Center Proceedings. No. 37. Washington, D.C., Department of Health, Education and Welfare Publication No. (NIH) 77-1063, 1977, pp. 73-82.

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DAVID F. SENIOR AND BIRDWELL FINLAYSON

12. Finlayson, B. : Renal lithiasis in review. Urol. Clin. North Am., 1:181-212, 1974. 13. Finlayson, B., and Roth, R.: Appraisal of calcium oxalate solubility in sodium chloride and sodium-calcium chloride solution. Urology, 1:142-144, 1973. 14. Hicking, W., Hesse, A., Gebhardt, M., et al.: Analytiche untersuchungen an harnsteinen von saugetieren. In Vahlensieck, W., and Gasser, G. (eds.): Harnstein Symposien Bonn-Wien. Darmstadt, Steinkopff Verlag, 1981, pp. 40-49. 15. Holtzapple, P. G., Bovee, K. C., Rea, C. F ., et al. : Amino acid uptake by kidney and jejunal tissue from dogs with cystine stones. Science, 166:1525-1527, 1969. 16. Khan, S. R., Finlayson, B., and Hackett, R. L.: Agar-embedded urinary stones: A technique useful for studying microscopic architecture. J. Urol., 130:992-995, 1983. 17. Mandel, N. S., and Mandel, G. S. : Epitaxis between stone-forming crystals at the atomic level. In Coe, F. L. (ed.): Nephrolithiasis. New York, Churchill Livingstone, 1980, pp. 37-58. 18. Moore, S., and Gowland, G.: The immunological integrity of matrix substance A and its possible detection and quantitation in urine. Br. J. Urol., 47:489-494, 1975. 19. Nancollas, G. H.: The kinetics of crystal growth and renal stone formation. In Fleisch, H., Robertson, W. G., Smith, L. H., et al. (eds.): Urolithiasis Research. New York, Plenum Press, 1976, pp. 5-23. 20. Pak, C. Y. C., Hayashi Y., Finlayson, B., et al. : Estimation of the state of saturation of brushite and calcium oxalate in urine: A comparison of three methods. J. Lab. Clin. Med., 89:891-901, 1977. 21. Parsons, C. L., Stauffer, C., Mulholland, S. G., et al.: Effect of ammonium on bacterial adherence to bladder transitional epithelium. J. Urol., 132:305-366, 1984. 22. Porter, P.: Colloidal properties ofurates in relation to calculus formation. Res. Vet. Sci., 7:128-137, 1966. 23. Porter, P.: Physico-chemical factors involved in urate calculus formation. I. Solubility. Res. Vet. Sci., 4:580-591, 1963. 24. Porter, P.: Physico-chemical factors involved in urate calculus formation. II. Colloidal flocculation. Res. Vet. Sci., 4:592-602, 1963. 25. Resnick, M. 1.: Urinary stone matrix. In Van Reen, R. (ed.): Idiopathic Urinary Stone Disease. Fogarty International Center Proceedings. No. 37. Washington, D.C., Department of Health, Education and Welfare Publication No. (NIH) 77-1063, 1977, pp. 73-82. 26. Robertson, W. G. : The solubility concept. In Nancollas, G. H., (ed.): Biological Mineralization and Demineralization. New York, Springer Verlag, 1982, pp. 5-21. 27. Robertson, W. G.: Saturation-inhibition index as a measure of the risk of calcium oxalate stone formation in the urinary tract. N. Engl. J. Med., 294:249-252, 1976. 28. Robertson, W. G., Peacock, M., and Nordin, B. E. C.: Activity products in stone-forming and non-stone-forming urine. Clin. Sci., 34:579-594, 1968. 29. Senior, D. F., Thomas, W. C., Jr., Gaskin, J. M., et al.: Relative merit of various nonsurgical treatments of infection stones in dogs. In Urolithiasis and Related Clinical Research. New York, Plenum Press, 1985, pp. 589-592. 30. Vermeulin, C. W., and Goetz, R.: Experimental urolithiasis. VIII. Furadantin in treatment of experimental proteus infection with stone formation. J. Urol., 72:99-104, 1954. 31. Wasserman, R. H., and Taylor, A. N.: Evidence of vitamin D-induced calcium-binding protein in primates. Proc. Soc. Exp. Bioi. Med., 136:25-28, 1971. 32. Weaver, A. D.: Canine urolithiasis. Incidence, chemical composition and outcome of 100 cases. J. Small Anim. Pract., 11:93-103, 1970. 33. White, E. G.: Symposium on urolithiasis. I. Introduction and incidences. J. Small Anim. Pract., 7:529-535, 1966. 34. Wickham, J. E. A.: The matrix of renal calculi. In Chisholm, G. D., and Williams, D. I. (eds.): Scientific Foundations of Urology. Edition 2. Chicago, Year Book Medical Publishers, Inc., 1982, pp. 323-329.

Department of Medical Sciences College of Veterinary Medicine University of Florida Gainesville, Florida 32601