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Origin and Types of Calcium Oxalate Monohydrate Papillary Renal Calculi
Endourology And Stones Origin and Types of Calcium Oxalate Monohydrate Papillary Renal Calculi Fèlix Grases, Antonia Costa-Bauzá, Isabel Gomila, and Antonio Conte OBJECTIVES
METHODS
RESULTS
CONCLUSIONS
Subepithelial hydroxyapatite calcification of renal papilla is thought to be involved in the formation of calcium oxalate monohydrate (COM) papillary calculi. To assess the mechanism of formation, we sought to correlate the fine structure of papillary renal calculi with specific pathophysiologic conditions and urinary alterations. The study included 831 COM papillary renal calculi with established fine inner structures. A total of 24 patients with chronic stone formation were randomly selected, and their urine was collected and analyzed. The case history and lifestyle habits of these patients were obtained. The 831 papillary calculi could be classified into 1 of 4 main groups. Type I included small calculi in which COM columnar crystals begin to develop in the concave zone in close contact with papillary tissue. Type II calculi contained a hydroxyapatite core located in or near the concave zone. Type III consisted of calculi that developed on the tip of the papillae and in the concave zone, containing hydroxyapatite, calcified tissue, and calcified tubules. Type IV consisted of papillary calculi in which the core, which is situated near, but not in, the concave zone, is formed by intergrown COM crystals and organic matter. Many factors, including urinary alterations (eg, hyperoxaluria), associated diseases (eg, hypertension, diabetes), and consumption or exposure to cytotoxic substances (eg, analgesic abuse) were associated with these types of calculi. Our findings have indicated that injury is the first cause of papillary COM calculus formation, with the location of the injury determining the morphology of the resulting calculus. UROLOGY 76: 1339 –1345, 2010. © 2010 Elsevier Inc.
bout 70% of urinary stones are formed of calcium oxalate, with about one half of these calculi having calcium oxalate monohydrate (COM) as the main component of their original crystalline phase and the other half having calcium oxalate dihydrate as the main component. Calcium oxalate dihydrate crystals are thermodynamically unstable and are slowly transformed to stable COM crystals when they come into contact with urine. Nevertheless, the crystalline fine inner structure of calculi initially constituted of calcium oxalate dihydrate crystals can be clearly identified by scanning electron microscopy.1 COM calculi can be divided into 2 clearly different types1: COM papillary calculi, with a detectable site of attachment to the renal papilla (approximately 13% of urinary stones) and COM calculi with no detectable site of attachment to the renal epithelium that develop in renal cavities (approximately 16% of urinary stones). Several studies have reported on the genesis of COM
A
papillary calculi since the first description of papillary calcifications and their possible role in the development of COM papillary calculi. The first report of a precalculus lesion in the renal papilla suggested that subepithelial hydroxyapatite calcification of the renal papilla becomes the nidus of COM papillary calculi, resulting from the disruption of the papillary epithelial layer by hydroxyapatite plaque.2 It is unclear, however, why and where these calcifications are initiated. In patients susceptible to the development of calcium renal calculi, plaque has been found to initiate in thin-loop basement membranes.3 In contrast, in patients who have undergone bypass surgery, these plaques form as intratubular hydroxyapatite crystals in the renal collecting ducts.4 However, not all papillary calculi are associated with these plaques.1,5 To better understand the mechanism of papillary renal calculi formation, we assessed the correlation between the fine structure and specific pathophysiologic conditions and main urinary alterations in patients.
From the Laboratory of Renal Lithiasis Research, Faculty of Sciences, Universitary Institute of Health Sciences Research, University of Balearic Islands, Palma de Mallorca, Spain; and Policlínica “Miramar,” Palma de Mallorca, Spain Reprint requests: Fèlix Grases, Ph.D., Laboratory of Renal Lithiasis Research, Universitary Institute of Health Science Research (IUNICS), University of Balearic Islands Ctra. de Valdemossa km 7.5, Palma de Mallorca 07122 Spain. E-mail: [email protected] Submitted: November 26, 2009; received (with revisions): February 9, 2010
MATERIAL AND METHODS
© 2010 Elsevier Inc. All Rights Reserved
Patients and Samples Our study included 831 papillary renal calculi from 708 patients. From the recently collected papillary calculi, 24 patients with chronic stone formation were randomly selected after calculi spontaneous expulsion, without any specific inclusion 0090-4295/10/$36.00 doi:10.1016/j.urology.2010.02.022
1339
criteria. The urine of these patients was collected and analyzed, and the case history and lifestyle habits of these patients were obtained.
Renal Calculi Studies The collected stones were dried, placed in sterile containers, and immediately examined by stereoscopic microscopy (Optomic, Madrid, Spain), infrared spectrometry (Infrared Spectroscope Bruker IFS 66, Bruker, Ettlingen, Germany), scanning electron microscopy (Hitachi S-3400N, Hitachi, Tokyo, Japan), and microanalysis by X-ray energy dispersion spectrometry (XFlash Detector 4010, Bruker AXS, Berlin, Germany).6 The COM papillary calculi were identified. A typical papillary COM stone consists of an eccentric core located near the concave zone, where it attaches to the papillae, and a radially striated convex peripheral layer.1,7 After direct examination of the external aspect of each stone by stereoscopic microscopy, each calculus was sectioned into 2 parts along a plane as near as possible to the geometric center. The internal structure and core were assessed by scanning electron microscopy and X-ray microanalysis (or infrared spectrometry), which were used to identify the microcomponents present in the core and to confirm the papillary origin by examination of the concave external cavity.
B
A C
500 μm D
Analysis of Urinary Samples All subjects were consuming an unregulated diet at urine collection and none of the stone formers was receiving any pharmacologic treatment. Serum was drawn and analyzed for creatinine, calcium, magnesium, phosphorous, citrate, oxalate, and uric acid. Patients with renal failure or infected urine were excluded. The urine was collected for 24 hours into sterile flasks containing thymol as a preservative and immediately refrigerated. After collection, the volume was recorded, and the samples were stored at ⫺20°C until they were assayed. A 2-hour urine collection was performed after overnight fasting, and the pH was immediately measured with a glass electrode (Crison pH meter). This urine was used only for pH evaluation, because these samples did not experience the pH changes due to precipitation processes (calcium salts) that can occur during 24hour storage. However, these pH measurements better represent the urinary basal pH, because they would be less affected by dietary factors. Normally, urine was collected in duplicate 1-2 months after stone passage/removal. If the analytic results were discordant, a third collection was performed to confirm the analytic findings. The calcium, magnesium, and phosphorous concentrations were measured by inductively coupled plasma atomic spectroscopy. Uric acid and creatinine were measured by Roche Modular Analytics with 11875426216 and 11875663216 reagents, respectively, and citrate and oxalate were measured using the R-Biopharm enzymatic test kits 10139076035 and 10755699035, respectively. The urinary biochemical parameters were considered potential lithogenic factors under abnormal conditions.1
Clinical History, Lifestyle, and Dietary Habits All patients provided a detailed clinical history and were interviewed about any family history of urolithiasis and their professional activity, with special attention to possible exposure to cytotoxic compounds, including pesticides, weed killers, disinfectants, cleaning products, paints, and fuels. 1340
Figure 1. Type I COM papillary calculi that corresponded to little calculi in which COM columnar crystals started their development directly in concave zone in close contact with papillary tissue. (A) Schematic representation of location of intrapapillary hydroxyapatite calcification (arrow). (B) Representative structure scheme. (C) Section of renal calculus. (D) Lateral subepithelial hydroxyapatite deposit (intraoperative endoscopic image) in patient with COM papillary stone formation.
RESULTS According to the fine inner microstructure of the calculi, the presence of minute substances in the core calculi and their location, and the etiologic factors that could be deduced from their macro- and microstructures, the 831 papillary calculi could be classified into 4 main groups. Type I COM papillary calculi consisted of small calculi in which COM columnar crystals began to develop in the concave zone in close contact with papillary tissue (Fig. 1). This type of calculus was associated with lateral subepithelial hydroxyapatite deposits (Fig. 1). Type II COM corresponded to calculi with a hydroxyapatite core located in or near the concave zone (Fig. 2A-C). Type III UROLOGY 76 (6), 2010
B
E
A
G
C
2 mm
2 mm
D
F
500 μm
H
500 μm Figure 2. COM papillary calculi that corresponded to calculi with hydroxyapatite core. In type II (A-D), core is located in concave zone or near concave zone. (A) Schematic representation of location of intrapapillary extruded hydroxyapatite calcification (arrow). (B) Representative structure scheme. (C) Section of renal calculus. (D) Detail of hydroxyapatite core located near point of attachment to papillae. In type III (E-H), calculi develop on papilla tip. In concave zone can be identified hydroxyapatite, calcified tissue, and calcified tubules. (E) Schematic representation of tip of calcified/necrosed papillae. (F) Representative structure scheme. (G) Section of renal calculus. (H) Detail of point of attachment to renal papillae, in which calcified renal tubules can be seen.
consisted of calculi that developed on the tip of the papillae and in the concave zone contained hydroxyapatite, calcified tissue, and calcified tubules (Fig. 2D-F). Finally, type IV consisted of papillary calculi in which the core, situated near the concave zone, was formed by intergrown COM crystals and organic matter (Fig. 3). Types I-III corresponded to papillary calculi linked to Randall’s plaque, and type IV developed by a different mechanism. Of the 831 papillary calculi we assessed, 655 (78.8%) were types I-III and 176 (21.1%) were type IV. Table 1 lists the urinary alterations, clinical information, and lifestyle habits that might be related to papillary injury and the subsequent development of papillary calculi in the 24 selected patients with stone formation. Many factors, including urinary alterations (eg, hyperoxaluria), associated diseases (eg, hypertension, diabetes), and the consumption or exposure to cytotoxic substances UROLOGY 76 (6), 2010
(eg, analgesic abuse), were associated with these types of calculi.
COMMENT We found that a large number of papillary renal calculi (about 80%) initially developed owing to subepithelial calcification known as Randall’s plaque. This calcification can be sufficiently large to erode the epithelium and directly contact urine, facilitating the growth of columnar COM crystals, with hydroxyapatite plaque incorporated into the papillary calculi (type II). If calcification occurs at the papillary tip, an important area is usually calcified, calcified tubules can be identified and hydroxyapatite is incorporated into the calculi (type III). Alternatively, the hydroxyapatite plaque might be located in a subepithelial position, inducing the growth of small pap1341
A
B
C
2 mm
D 500 μm
Figure 3. Type IV COM papillary calculi in which the core, situated near the concave zone, is formed by intergrown COM crystals and organic matter. (A) Schematic representation of location of injury to outer epithelial layer that covers renal papillae (arrow). (B) Representative structure scheme. (C) Section of renal calculus. (D) Detail of core formed by intergrown COM crystals and organic matter.
illary calculi, such that, when the calculus becomes unattached, plaque does not appear and the epithelium separates the interstitial plaque from the urine and hydroxyapatite is not incorporated into the papillary calculi (type I). Owing to the absence of hydroxyapatite in the type I calculi and the presence of columnar COM crystals directly formed from the concave zone, to investigate the origin of type I calculi, the endoscopic image of the papillae was obtained after calculus expulsion. These situations were clearly described by Evan et al.8 Finally, in other cases, the papillary calculi showed a clearly developed core from which hydroxyapatite was absent. These cases contained intergrown COM crystals, organic matter, and other components, such as sodium urate (type IV). We found that about 20% of papillary calculi 1342
had type IV characteristics, greater than the 10% reported previously.5 Human kidneys have been reported to have different regions of tissue calcification, including coarse focal deposits in the papillary region and calcifications around the loops of Henle, which can be seen in patients of all ages. Calcifications present at the boundary of the inner and outer medullary regions have been associated with degenerative changes, aging, and arteriolar disease.3,9 The development of these calcified areas seems to follow an injury or a pre-existing lesion, such as papillary necrosis, which leads to intratubular calcium phosphate deposits.10 Papillary calcification can be induced in rats with a combination of aspirin and sodium saccharin,11,12 and calcification of the vasa recta can be induced by long-term phenacetin.13 In addition, high doses of phenylbutazone, oxyphenbutazone, and indomethacin in rats has led to tubular necrosis in the lower nephron, causing calcification.14 Renal papillary necrosis and calcification have also developed in patients with diabetes mellitus.15 Hyperoxaluria, which leads to hydroxyapatite tubular deposits in the lumen of collecting ducts and calcium oxalate monohydrate crystal deposits on the papillary tips, is a precursor to papillary stones.4,16,17 High-resolution radiography to identify Randall’s plaques in cadaveric kidneys found medullary calcifications in 57% of the renal units.18 Calcium deposits were localized to the basement membrane of collecting tubules and the vasa recta and to the papillary interstitium. A history of hypertension was the only clinical parameter correlating with papillary calcifications. In other cases, the nidus might have occurred at sites with altered (damaged or just slightly injured) epithelium.19,20 Although the development of tissue calcification depends on a pre-existing injury, which acts as an inducer, the continuation of this process is subject to modulators and/or a deficiency in crystallization inhibitors. For example, some carboxy proteins, such as osteopontin, can bind hydroxyapatite and thus recruit macrophages that remove these calcifications or prevent their progression.21-26 The inhibition of crystallization, avoiding hydroxyapatite development (nucleation and growth), has been attributed to low-molecular-weight compounds, such as pyrophosphate, magnesium, and phytate.27-29 We found the presence of systemic diseases, hyperoxaluria, and hyperuricemia to be possible causes of papillary injury and the formation of hydroxyapatite deposits. In addition, exposure to metals (eg, mercury, lithium), pesticides, or food additives, which have nephrotoxic activity, might induce intrapapillary calcifications. The marked increase in calcium oxalate papillary lithiasis5 might result from these circumstances and warrants additional study. It would be instructive to prospectively collect data from on nonstone-forming patients undergoing ureteroscopy (for nonstone disease) but with attention to analogous lifestyle/exposure to know whether such patients have similar evidence of renal injury but UROLOGY 76 (6), 2010
Table 1. Urinary Alterations, Clinical Information, and Other Relevant Factors* Related to Papillary Injury and Development of Papillary Calculi of 24 Selected Patients with Stone Formation Age (y)/ Sex
Pt. No.
Papillary Calculi Type
1
I
44/
No
—
First
Migraine headache
2
II
46/
No
Low phytate consumption
First
—
Rizatriptan (analgesic antimigraine) —
3
II
70
No
First
—
—
4
I
17
Low phytate consumption, smoker (30-40 cigarettes/ d) exposure to HCl, Cl2 Yes Low phytate (parents) consumption
First
—
—
5
I
66
Yes (sister)
Previous episodes
Diabetes
—
6
IV
54
Yes — (mother)
First
—
7
III
37/F
No
—
First
Depression
8
IV
39/M
No
First
—
9 10
I I
41/M 59/M
No No
Low phytate consumption — Exsmoker
Acetaminophen (analgesic), sertraline (antidepressant) Fluoxetine (antidepressant) —
First Previous episodes
— —
11
I
59/M
No
—
Previous episodes
Hypertension Diabetes, chronic headache, silicosis (coal exposure) —
12
IV
78/M
No
First
Hypertension
—
13
III
38/F
Yes
Low phytate consumption —
Migraine headache
14
I
45/F
No
—
Previous episodes First
Acetaminophen (analgesic) —
15
IV
60/F
Yes
Exposure to HCl, Cl2
Previous episodes
16
I
29/F
Yes
—
First
17
II
34/M
Yes
—
18
II
23/M
No
Low phytate consumption
UROLOGY 76 (6), 2010
Family History
Lifestyle Habits
—
Episode
Chronic Disease
Overweight, hyperparathyroidism
Chronic Drug Consumption
—
Gastroduodenal ulcer, gastroduodenal bleeding Hyperreactivity of bronchial epithelium
—
Previous episodes
Gastroduodenal ulcer
—
Previous episodes
—
—
—
Urinary/Plasmatic Biochemical Alterations† High urinary calcium (211 mg/L), urinary pH 6.5 Hyperuricemia (7.8mg/ dL), high urinary oxalate (38 mg/L), urinary pH 7, low urinary citrate (250mg/L) Hyperuricemia (7.3mg/ dL), high urinary oxalate (46 mg/L), low urinary citrate (250 mg/L)
High urinary urate (904 mg/L), high urinary calcium (242 mg/L), high urinary oxalate (44 mg/L) High urinary urate (570 mg/L), high urinary calcium (210mg/L), high urinary oxalate (41 mg/L), urinary pH 6.5 —
High urinary oxalate (36 mg/L) High urinary oxalate (46 mg/L) Urinary pH 5 Hyperuricemia, hypercholesterolemia
High urinary urate (540 mg/L), high urinary oxalate (30 mg/L), urinary pH 5.0 Hyperuricemia, hypercholesterolemia Hypercholesterolemia High urinary calcium (215 mg/L), high urinary oxalate (33mg/L) —
High urinary urate (703 mg/L), high urinary calcium (321 mg/L), high urinary oxalate (30 mg/L) High urinary calcium (278 mg/L), low urinary citrate (200 mg/L), urinary pH ⬎6 High urinary oxalate (33 mg/L) Continued
1343
Table 1. Continued Pt. No.
Papillary Calculi Type
Age (y)/ Sex
Family History
19
I
34/M
Yes
Low phytate consumption
Previous episodes
Diabetes
—
20
I
31/M
Yes
—
First
—
—
21
I
60/M
Yes
—
Previous episodes
22
I
50/M
Yes
—
Antiinflammatory treatment —
23
IV
50/M
No
24
II
55/M
No
High tea consumption —
Previous episodes First
Hypertension, arthritis, dyslipidemia Hypertension, migraine Gastroduodenal ulcer Hypertension, migraine
Lifestyle Habits
Episode
First
Chronic Disease
Chronic Drug Consumption
— Acetaminophen (analgesic)
Urinary/Plasmatic Biochemical Alterations† High urinary urate (730 mg/L), high urinary calcium (338 mg/L), urinary pH 5.0 Hyperuricemia, high urinary oxalate (32 mg/L), urinary pH 7 High urinary calcium (220 mg/L) High urinary calcium (254 mg/L) pH 5 —
Pt. No., patient number. * Chronic diseases, chronic consumption of drugs, exposure to cytotoxic agents. † Urinary biochemical data presented as mean of 2 different analyses.
fail to grow clinical stones because of more favorable urine chemistry or whether they just do not have renal injury from the outset. Not all intrapapillary calcifications can induce the formation of papillary COM calculi. Although close contact between urine and the plaque is required, some hydroxyapatite deposits are not in contact with urine and therefore will not be involved in the development of calculi. An evaluation of Randall’s plaque theory of nephrolithiasis using computed tomography attenuation values (Hounsfield units) in patients with unilateral single calculi found that the Hounsfield units of all papillae on the affected side was significantly greater in those with stone formation than in the control group.30 In contrast, no significant differences were found between the affected and nonaffected sides of the patients with stone formation.30
CONCLUSIONS Our findings have indicated that an injury is the first cause of papillary COM calculi formation. An injury to the outer epithelial layer that covers the renal papillae, followed by the inability of crystallization inhibitors and/or the immune system to halt this process, can lead the supersaturated components of urine (mainly calcium oxalate) to form a nidus on the papillae (constituting the core of the calculus), with columnar COM crystals constituting the main body of these calculi. Alternatively, an injury inside the tissue of the renal papillae, followed by the inability of crystallization inhibitors and/or the immune system to halt this process, can lead to the formation of a hydroxyapatite plaque. If these calcified plaques come into close contact with urine, COM columnar crystals will grow, resulting in the formation of a papillary COM calculus. Hence, the identification of the cause or causes of the injury responsible for papillary COM cal1344
culus formation is central to the design of adequate prophylactic measures. Acknowledgment. To the Dirección de Investigación (Proyecto CTQ2006-05640) and Gobierno de las Islas Baleares (PCTIB-2005GC4-06) for their financial support; and to Conselleria d’Innovació I Energia Del Govern de Les Illes Balears for a fellowship to I. Gomila. References 1. Grases F, Costa-Bauzá A, Ramis M, et al. Simple classification of renal calculi closely related to their micromorphology and etiology. Clin Chim Acta. 2002;322:29-36. 2. Prien EL. The riddle of Randall’s plaques. J Urol. 1975;114:500507. 3. Evan AP, Lingeman JE, Coe FL, et al. Randall’s plaque of patients with nephrolithiasis begins in basement membranes of thin loops of Henle. J Clin Invest. 2003;111:607-616. 4. O’Connor RC, Worcester EM, Evan AP, et al. Nephrolithiasis and nephrocalcinosis in rats with small bowel resection. Urol Res. 2005;33:105-115. 5. Daudon M, Carpentier X, Traxer O, et al. Randall’s plaque: an increasingly frequent and complex process in calcium stone formation. Urol Res. 2008;36:162. 6. Grases F, García-Ferragut L, Costa-Bauzá A. Analytical study of renal calculi: a new insight. Recent Res Dev Pure Appl Anal Chem. 1998;1:187-206. 7. Grases F, Costa-Bauzá A, Garcia-Ferragut L. Biopathological crystallization: a general view about the mechanisms of renal stone formation. Adv Colloid Interface Sci. 1998;74:169-194. 8. Evan AP, Coe FL, Lingeman JE, et al. Mechanism of formation of human calcium oxalate renal stones on Randall’s plaque. Anat Rec. 2007;290:1315-1323. 9. Evan AP, Coe FL, Rittling SR, et al. Apatite plaque particles in inner medulla of kidneys of calcium oxalate stone formers: osteopontin localization. Kidney Int. 2005;68:145-154. 10. Hennis HL, Hennigar GR, Greene WB, et al. Intratubular calcium phosphate deposition in acute analgesic nephropathy in rabbits. Am J Pathol. 1982;106:356-363. 11. Johansson SL, Sakata T, Hasegawa R, et al. The effect of long-term administration of aspirin and sodium saccharin on the rat kidney. Toxicol Appl Pharmacol. 1986;86:80-92.
UROLOGY 76 (6), 2010
12. Molland EA. Aspirin damage in the rat kidney in the intact animal and after unilateral nephrectomy. J Pathol. 1976;120:43-48. 13. Johansson S, Angervall L. Urothelial changes of the renal papillae in Sprague-Dawley rats induced by long term feeding of phenacetin. Acta Pathol Microbiol Scand. 1976;84:375-383. 14. Arnold L, Collins C, Starmer GA. Further studies of the acute effects of phenylbutazone, oxyphenbutazone and indomethacin on the rat kidney. Pathology. 1976;8:135-141. 15. Ellenbogen PH, Talner LB. Uroradiology of diabetes mellitus. Urology. 1976;8:413-419. 16. Di Tommaso L, Tolomelli B, Mezzini R, et al. Renal calcium phosphate and oxalate deposition in prolonged vitamin B6 deficiency: studies on a rat model of urolithiasis. BJU Int. 2002;89:571575. 17. Mandel NS, Henderson JD Jr, Hung LY, et al. A porcine model of calcium oxalate kidney stone disease. J Urol. 2004;171:1301-1303. 18. Stoller M, Low R, Shami G, et al. High resolution radiography if cadaveric kidneys: unraveling the mystery of Randall’s plaque formation. J Urol. 1996;156:1263-1266. 19. Gill WB, Jones KW, Ruggiero KJ. Protective effects of heparin and other sulfated glycosaminoglycans on crystal adhesion to injured urothelium. J Urol. 1982;127:152-154. 20. Lieske JC, Norris R, Swift H, et al. Adhesion, internalization and metabolism of calcium oxalate monohydrate crystals by renal epithelial cells. Kidney Int. 1997;52:1291-1301. 21. Steitz SA, Speer MY, McKee MD, et al. Osteopontin inhibits mineral deposition and promotes regression of ectopic calcification. Am J Pathol. 2002;161:2035-2046.
UROLOGY 76 (6), 2010
22. Romberg RW, Werness PG, Riggs BL, et al. Inhibition of hydroxyapatite crystal growth by bone-specific and other calcium-binding proteins. Biochemistry. 1986;25:1176-1180. 23. Boskey AL, Maresca M, Ullrich W, et al. Osteopontin-hydroxyapatite interactions in vitro: inhibition of hydroxyapatite formation and growth in a gelatin-gel. Bone Miner. 1993;22:147-159. 24. Govindaraj A, Selvam R. An oxalate-binding protein with crystal growth promoter activity from human kidney stone matrix. BJU Int. 2002;90:336-344. 25. Yamate T, Kohri K, Umekawa T, et al. The effect of osteopontin on the adhesion of calcium oxalate crystals to Madin-Darby canine kidney cells. Eur Urol. 1996;30:388-393. 26. Lieske JC, Toback FG, Deganello S. Sialic acid-containing glycoproteins on renal cells determine nucleation of calcium oxalate dihydrate crystals. Kidney Int. 2001;60:1784-1791. 27. Lomashvili KA, Cobbs S, Hennigar RA, et al. Phosphate-induced vascular calcification: role of pyrophosphate and osteopontin. J Am Soc Nephrol. 2004;15:1392-1401. 28. Wilson JW, Werness PG, Smith LH. Inhibitors of crystal growth of hydroxyapatite: a constant composition approach. J Urol. 1985;134: 1255-1258. 29. Grases F, Ramis M, Costa-Bauzá A. Effects of phytate and pyrophosphate on brushite and hydroxyapatite crystallization: comparison with the action of other polyphosphates. Urol Res. 2000;28: 136-140. 30. Bhuskute NM, Yap WW, Wah TM. A retrospective evaluation of Randall’s plaque theory of nephrolithiasis with CT attenuation values. Eur J Radiol. 2009;72:470-472.
1345
METHODS
RESULTS
CONCLUSIONS
Subepithelial hydroxyapatite calcification of renal papilla is thought to be involved in the formation of calcium oxalate monohydrate (COM) papillary calculi. To assess the mechanism of formation, we sought to correlate the fine structure of papillary renal calculi with specific pathophysiologic conditions and urinary alterations. The study included 831 COM papillary renal calculi with established fine inner structures. A total of 24 patients with chronic stone formation were randomly selected, and their urine was collected and analyzed. The case history and lifestyle habits of these patients were obtained. The 831 papillary calculi could be classified into 1 of 4 main groups. Type I included small calculi in which COM columnar crystals begin to develop in the concave zone in close contact with papillary tissue. Type II calculi contained a hydroxyapatite core located in or near the concave zone. Type III consisted of calculi that developed on the tip of the papillae and in the concave zone, containing hydroxyapatite, calcified tissue, and calcified tubules. Type IV consisted of papillary calculi in which the core, which is situated near, but not in, the concave zone, is formed by intergrown COM crystals and organic matter. Many factors, including urinary alterations (eg, hyperoxaluria), associated diseases (eg, hypertension, diabetes), and consumption or exposure to cytotoxic substances (eg, analgesic abuse) were associated with these types of calculi. Our findings have indicated that injury is the first cause of papillary COM calculus formation, with the location of the injury determining the morphology of the resulting calculus. UROLOGY 76: 1339 –1345, 2010. © 2010 Elsevier Inc.
bout 70% of urinary stones are formed of calcium oxalate, with about one half of these calculi having calcium oxalate monohydrate (COM) as the main component of their original crystalline phase and the other half having calcium oxalate dihydrate as the main component. Calcium oxalate dihydrate crystals are thermodynamically unstable and are slowly transformed to stable COM crystals when they come into contact with urine. Nevertheless, the crystalline fine inner structure of calculi initially constituted of calcium oxalate dihydrate crystals can be clearly identified by scanning electron microscopy.1 COM calculi can be divided into 2 clearly different types1: COM papillary calculi, with a detectable site of attachment to the renal papilla (approximately 13% of urinary stones) and COM calculi with no detectable site of attachment to the renal epithelium that develop in renal cavities (approximately 16% of urinary stones). Several studies have reported on the genesis of COM
A
papillary calculi since the first description of papillary calcifications and their possible role in the development of COM papillary calculi. The first report of a precalculus lesion in the renal papilla suggested that subepithelial hydroxyapatite calcification of the renal papilla becomes the nidus of COM papillary calculi, resulting from the disruption of the papillary epithelial layer by hydroxyapatite plaque.2 It is unclear, however, why and where these calcifications are initiated. In patients susceptible to the development of calcium renal calculi, plaque has been found to initiate in thin-loop basement membranes.3 In contrast, in patients who have undergone bypass surgery, these plaques form as intratubular hydroxyapatite crystals in the renal collecting ducts.4 However, not all papillary calculi are associated with these plaques.1,5 To better understand the mechanism of papillary renal calculi formation, we assessed the correlation between the fine structure and specific pathophysiologic conditions and main urinary alterations in patients.
From the Laboratory of Renal Lithiasis Research, Faculty of Sciences, Universitary Institute of Health Sciences Research, University of Balearic Islands, Palma de Mallorca, Spain; and Policlínica “Miramar,” Palma de Mallorca, Spain Reprint requests: Fèlix Grases, Ph.D., Laboratory of Renal Lithiasis Research, Universitary Institute of Health Science Research (IUNICS), University of Balearic Islands Ctra. de Valdemossa km 7.5, Palma de Mallorca 07122 Spain. E-mail: [email protected] Submitted: November 26, 2009; received (with revisions): February 9, 2010
MATERIAL AND METHODS
© 2010 Elsevier Inc. All Rights Reserved
Patients and Samples Our study included 831 papillary renal calculi from 708 patients. From the recently collected papillary calculi, 24 patients with chronic stone formation were randomly selected after calculi spontaneous expulsion, without any specific inclusion 0090-4295/10/$36.00 doi:10.1016/j.urology.2010.02.022
1339
criteria. The urine of these patients was collected and analyzed, and the case history and lifestyle habits of these patients were obtained.
Renal Calculi Studies The collected stones were dried, placed in sterile containers, and immediately examined by stereoscopic microscopy (Optomic, Madrid, Spain), infrared spectrometry (Infrared Spectroscope Bruker IFS 66, Bruker, Ettlingen, Germany), scanning electron microscopy (Hitachi S-3400N, Hitachi, Tokyo, Japan), and microanalysis by X-ray energy dispersion spectrometry (XFlash Detector 4010, Bruker AXS, Berlin, Germany).6 The COM papillary calculi were identified. A typical papillary COM stone consists of an eccentric core located near the concave zone, where it attaches to the papillae, and a radially striated convex peripheral layer.1,7 After direct examination of the external aspect of each stone by stereoscopic microscopy, each calculus was sectioned into 2 parts along a plane as near as possible to the geometric center. The internal structure and core were assessed by scanning electron microscopy and X-ray microanalysis (or infrared spectrometry), which were used to identify the microcomponents present in the core and to confirm the papillary origin by examination of the concave external cavity.
B
A C
500 μm D
Analysis of Urinary Samples All subjects were consuming an unregulated diet at urine collection and none of the stone formers was receiving any pharmacologic treatment. Serum was drawn and analyzed for creatinine, calcium, magnesium, phosphorous, citrate, oxalate, and uric acid. Patients with renal failure or infected urine were excluded. The urine was collected for 24 hours into sterile flasks containing thymol as a preservative and immediately refrigerated. After collection, the volume was recorded, and the samples were stored at ⫺20°C until they were assayed. A 2-hour urine collection was performed after overnight fasting, and the pH was immediately measured with a glass electrode (Crison pH meter). This urine was used only for pH evaluation, because these samples did not experience the pH changes due to precipitation processes (calcium salts) that can occur during 24hour storage. However, these pH measurements better represent the urinary basal pH, because they would be less affected by dietary factors. Normally, urine was collected in duplicate 1-2 months after stone passage/removal. If the analytic results were discordant, a third collection was performed to confirm the analytic findings. The calcium, magnesium, and phosphorous concentrations were measured by inductively coupled plasma atomic spectroscopy. Uric acid and creatinine were measured by Roche Modular Analytics with 11875426216 and 11875663216 reagents, respectively, and citrate and oxalate were measured using the R-Biopharm enzymatic test kits 10139076035 and 10755699035, respectively. The urinary biochemical parameters were considered potential lithogenic factors under abnormal conditions.1
Clinical History, Lifestyle, and Dietary Habits All patients provided a detailed clinical history and were interviewed about any family history of urolithiasis and their professional activity, with special attention to possible exposure to cytotoxic compounds, including pesticides, weed killers, disinfectants, cleaning products, paints, and fuels. 1340
Figure 1. Type I COM papillary calculi that corresponded to little calculi in which COM columnar crystals started their development directly in concave zone in close contact with papillary tissue. (A) Schematic representation of location of intrapapillary hydroxyapatite calcification (arrow). (B) Representative structure scheme. (C) Section of renal calculus. (D) Lateral subepithelial hydroxyapatite deposit (intraoperative endoscopic image) in patient with COM papillary stone formation.
RESULTS According to the fine inner microstructure of the calculi, the presence of minute substances in the core calculi and their location, and the etiologic factors that could be deduced from their macro- and microstructures, the 831 papillary calculi could be classified into 4 main groups. Type I COM papillary calculi consisted of small calculi in which COM columnar crystals began to develop in the concave zone in close contact with papillary tissue (Fig. 1). This type of calculus was associated with lateral subepithelial hydroxyapatite deposits (Fig. 1). Type II COM corresponded to calculi with a hydroxyapatite core located in or near the concave zone (Fig. 2A-C). Type III UROLOGY 76 (6), 2010
B
E
A
G
C
2 mm
2 mm
D
F
500 μm
H
500 μm Figure 2. COM papillary calculi that corresponded to calculi with hydroxyapatite core. In type II (A-D), core is located in concave zone or near concave zone. (A) Schematic representation of location of intrapapillary extruded hydroxyapatite calcification (arrow). (B) Representative structure scheme. (C) Section of renal calculus. (D) Detail of hydroxyapatite core located near point of attachment to papillae. In type III (E-H), calculi develop on papilla tip. In concave zone can be identified hydroxyapatite, calcified tissue, and calcified tubules. (E) Schematic representation of tip of calcified/necrosed papillae. (F) Representative structure scheme. (G) Section of renal calculus. (H) Detail of point of attachment to renal papillae, in which calcified renal tubules can be seen.
consisted of calculi that developed on the tip of the papillae and in the concave zone contained hydroxyapatite, calcified tissue, and calcified tubules (Fig. 2D-F). Finally, type IV consisted of papillary calculi in which the core, situated near the concave zone, was formed by intergrown COM crystals and organic matter (Fig. 3). Types I-III corresponded to papillary calculi linked to Randall’s plaque, and type IV developed by a different mechanism. Of the 831 papillary calculi we assessed, 655 (78.8%) were types I-III and 176 (21.1%) were type IV. Table 1 lists the urinary alterations, clinical information, and lifestyle habits that might be related to papillary injury and the subsequent development of papillary calculi in the 24 selected patients with stone formation. Many factors, including urinary alterations (eg, hyperoxaluria), associated diseases (eg, hypertension, diabetes), and the consumption or exposure to cytotoxic substances UROLOGY 76 (6), 2010
(eg, analgesic abuse), were associated with these types of calculi.
COMMENT We found that a large number of papillary renal calculi (about 80%) initially developed owing to subepithelial calcification known as Randall’s plaque. This calcification can be sufficiently large to erode the epithelium and directly contact urine, facilitating the growth of columnar COM crystals, with hydroxyapatite plaque incorporated into the papillary calculi (type II). If calcification occurs at the papillary tip, an important area is usually calcified, calcified tubules can be identified and hydroxyapatite is incorporated into the calculi (type III). Alternatively, the hydroxyapatite plaque might be located in a subepithelial position, inducing the growth of small pap1341
A
B
C
2 mm
D 500 μm
Figure 3. Type IV COM papillary calculi in which the core, situated near the concave zone, is formed by intergrown COM crystals and organic matter. (A) Schematic representation of location of injury to outer epithelial layer that covers renal papillae (arrow). (B) Representative structure scheme. (C) Section of renal calculus. (D) Detail of core formed by intergrown COM crystals and organic matter.
illary calculi, such that, when the calculus becomes unattached, plaque does not appear and the epithelium separates the interstitial plaque from the urine and hydroxyapatite is not incorporated into the papillary calculi (type I). Owing to the absence of hydroxyapatite in the type I calculi and the presence of columnar COM crystals directly formed from the concave zone, to investigate the origin of type I calculi, the endoscopic image of the papillae was obtained after calculus expulsion. These situations were clearly described by Evan et al.8 Finally, in other cases, the papillary calculi showed a clearly developed core from which hydroxyapatite was absent. These cases contained intergrown COM crystals, organic matter, and other components, such as sodium urate (type IV). We found that about 20% of papillary calculi 1342
had type IV characteristics, greater than the 10% reported previously.5 Human kidneys have been reported to have different regions of tissue calcification, including coarse focal deposits in the papillary region and calcifications around the loops of Henle, which can be seen in patients of all ages. Calcifications present at the boundary of the inner and outer medullary regions have been associated with degenerative changes, aging, and arteriolar disease.3,9 The development of these calcified areas seems to follow an injury or a pre-existing lesion, such as papillary necrosis, which leads to intratubular calcium phosphate deposits.10 Papillary calcification can be induced in rats with a combination of aspirin and sodium saccharin,11,12 and calcification of the vasa recta can be induced by long-term phenacetin.13 In addition, high doses of phenylbutazone, oxyphenbutazone, and indomethacin in rats has led to tubular necrosis in the lower nephron, causing calcification.14 Renal papillary necrosis and calcification have also developed in patients with diabetes mellitus.15 Hyperoxaluria, which leads to hydroxyapatite tubular deposits in the lumen of collecting ducts and calcium oxalate monohydrate crystal deposits on the papillary tips, is a precursor to papillary stones.4,16,17 High-resolution radiography to identify Randall’s plaques in cadaveric kidneys found medullary calcifications in 57% of the renal units.18 Calcium deposits were localized to the basement membrane of collecting tubules and the vasa recta and to the papillary interstitium. A history of hypertension was the only clinical parameter correlating with papillary calcifications. In other cases, the nidus might have occurred at sites with altered (damaged or just slightly injured) epithelium.19,20 Although the development of tissue calcification depends on a pre-existing injury, which acts as an inducer, the continuation of this process is subject to modulators and/or a deficiency in crystallization inhibitors. For example, some carboxy proteins, such as osteopontin, can bind hydroxyapatite and thus recruit macrophages that remove these calcifications or prevent their progression.21-26 The inhibition of crystallization, avoiding hydroxyapatite development (nucleation and growth), has been attributed to low-molecular-weight compounds, such as pyrophosphate, magnesium, and phytate.27-29 We found the presence of systemic diseases, hyperoxaluria, and hyperuricemia to be possible causes of papillary injury and the formation of hydroxyapatite deposits. In addition, exposure to metals (eg, mercury, lithium), pesticides, or food additives, which have nephrotoxic activity, might induce intrapapillary calcifications. The marked increase in calcium oxalate papillary lithiasis5 might result from these circumstances and warrants additional study. It would be instructive to prospectively collect data from on nonstone-forming patients undergoing ureteroscopy (for nonstone disease) but with attention to analogous lifestyle/exposure to know whether such patients have similar evidence of renal injury but UROLOGY 76 (6), 2010
Table 1. Urinary Alterations, Clinical Information, and Other Relevant Factors* Related to Papillary Injury and Development of Papillary Calculi of 24 Selected Patients with Stone Formation Age (y)/ Sex
Pt. No.
Papillary Calculi Type
1
I
44/
No
—
First
Migraine headache
2
II
46/
No
Low phytate consumption
First
—
Rizatriptan (analgesic antimigraine) —
3
II
70
No
First
—
—
4
I
17
Low phytate consumption, smoker (30-40 cigarettes/ d) exposure to HCl, Cl2 Yes Low phytate (parents) consumption
First
—
—
5
I
66
Yes (sister)
Previous episodes
Diabetes
—
6
IV
54
Yes — (mother)
First
—
7
III
37/F
No
—
First
Depression
8
IV
39/M
No
First
—
9 10
I I
41/M 59/M
No No
Low phytate consumption — Exsmoker
Acetaminophen (analgesic), sertraline (antidepressant) Fluoxetine (antidepressant) —
First Previous episodes
— —
11
I
59/M
No
—
Previous episodes
Hypertension Diabetes, chronic headache, silicosis (coal exposure) —
12
IV
78/M
No
First
Hypertension
—
13
III
38/F
Yes
Low phytate consumption —
Migraine headache
14
I
45/F
No
—
Previous episodes First
Acetaminophen (analgesic) —
15
IV
60/F
Yes
Exposure to HCl, Cl2
Previous episodes
16
I
29/F
Yes
—
First
17
II
34/M
Yes
—
18
II
23/M
No
Low phytate consumption
UROLOGY 76 (6), 2010
Family History
Lifestyle Habits
—
Episode
Chronic Disease
Overweight, hyperparathyroidism
Chronic Drug Consumption
—
Gastroduodenal ulcer, gastroduodenal bleeding Hyperreactivity of bronchial epithelium
—
Previous episodes
Gastroduodenal ulcer
—
Previous episodes
—
—
—
Urinary/Plasmatic Biochemical Alterations† High urinary calcium (211 mg/L), urinary pH 6.5 Hyperuricemia (7.8mg/ dL), high urinary oxalate (38 mg/L), urinary pH 7, low urinary citrate (250mg/L) Hyperuricemia (7.3mg/ dL), high urinary oxalate (46 mg/L), low urinary citrate (250 mg/L)
High urinary urate (904 mg/L), high urinary calcium (242 mg/L), high urinary oxalate (44 mg/L) High urinary urate (570 mg/L), high urinary calcium (210mg/L), high urinary oxalate (41 mg/L), urinary pH 6.5 —
High urinary oxalate (36 mg/L) High urinary oxalate (46 mg/L) Urinary pH 5 Hyperuricemia, hypercholesterolemia
High urinary urate (540 mg/L), high urinary oxalate (30 mg/L), urinary pH 5.0 Hyperuricemia, hypercholesterolemia Hypercholesterolemia High urinary calcium (215 mg/L), high urinary oxalate (33mg/L) —
High urinary urate (703 mg/L), high urinary calcium (321 mg/L), high urinary oxalate (30 mg/L) High urinary calcium (278 mg/L), low urinary citrate (200 mg/L), urinary pH ⬎6 High urinary oxalate (33 mg/L) Continued
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Table 1. Continued Pt. No.
Papillary Calculi Type
Age (y)/ Sex
Family History
19
I
34/M
Yes
Low phytate consumption
Previous episodes
Diabetes
—
20
I
31/M
Yes
—
First
—
—
21
I
60/M
Yes
—
Previous episodes
22
I
50/M
Yes
—
Antiinflammatory treatment —
23
IV
50/M
No
24
II
55/M
No
High tea consumption —
Previous episodes First
Hypertension, arthritis, dyslipidemia Hypertension, migraine Gastroduodenal ulcer Hypertension, migraine
Lifestyle Habits
Episode
First
Chronic Disease
Chronic Drug Consumption
— Acetaminophen (analgesic)
Urinary/Plasmatic Biochemical Alterations† High urinary urate (730 mg/L), high urinary calcium (338 mg/L), urinary pH 5.0 Hyperuricemia, high urinary oxalate (32 mg/L), urinary pH 7 High urinary calcium (220 mg/L) High urinary calcium (254 mg/L) pH 5 —
Pt. No., patient number. * Chronic diseases, chronic consumption of drugs, exposure to cytotoxic agents. † Urinary biochemical data presented as mean of 2 different analyses.
fail to grow clinical stones because of more favorable urine chemistry or whether they just do not have renal injury from the outset. Not all intrapapillary calcifications can induce the formation of papillary COM calculi. Although close contact between urine and the plaque is required, some hydroxyapatite deposits are not in contact with urine and therefore will not be involved in the development of calculi. An evaluation of Randall’s plaque theory of nephrolithiasis using computed tomography attenuation values (Hounsfield units) in patients with unilateral single calculi found that the Hounsfield units of all papillae on the affected side was significantly greater in those with stone formation than in the control group.30 In contrast, no significant differences were found between the affected and nonaffected sides of the patients with stone formation.30
CONCLUSIONS Our findings have indicated that an injury is the first cause of papillary COM calculi formation. An injury to the outer epithelial layer that covers the renal papillae, followed by the inability of crystallization inhibitors and/or the immune system to halt this process, can lead the supersaturated components of urine (mainly calcium oxalate) to form a nidus on the papillae (constituting the core of the calculus), with columnar COM crystals constituting the main body of these calculi. Alternatively, an injury inside the tissue of the renal papillae, followed by the inability of crystallization inhibitors and/or the immune system to halt this process, can lead to the formation of a hydroxyapatite plaque. If these calcified plaques come into close contact with urine, COM columnar crystals will grow, resulting in the formation of a papillary COM calculus. Hence, the identification of the cause or causes of the injury responsible for papillary COM cal1344
culus formation is central to the design of adequate prophylactic measures. Acknowledgment. To the Dirección de Investigación (Proyecto CTQ2006-05640) and Gobierno de las Islas Baleares (PCTIB-2005GC4-06) for their financial support; and to Conselleria d’Innovació I Energia Del Govern de Les Illes Balears for a fellowship to I. Gomila. References 1. Grases F, Costa-Bauzá A, Ramis M, et al. Simple classification of renal calculi closely related to their micromorphology and etiology. Clin Chim Acta. 2002;322:29-36. 2. Prien EL. The riddle of Randall’s plaques. J Urol. 1975;114:500507. 3. Evan AP, Lingeman JE, Coe FL, et al. Randall’s plaque of patients with nephrolithiasis begins in basement membranes of thin loops of Henle. J Clin Invest. 2003;111:607-616. 4. O’Connor RC, Worcester EM, Evan AP, et al. Nephrolithiasis and nephrocalcinosis in rats with small bowel resection. Urol Res. 2005;33:105-115. 5. Daudon M, Carpentier X, Traxer O, et al. Randall’s plaque: an increasingly frequent and complex process in calcium stone formation. Urol Res. 2008;36:162. 6. Grases F, García-Ferragut L, Costa-Bauzá A. Analytical study of renal calculi: a new insight. Recent Res Dev Pure Appl Anal Chem. 1998;1:187-206. 7. Grases F, Costa-Bauzá A, Garcia-Ferragut L. Biopathological crystallization: a general view about the mechanisms of renal stone formation. Adv Colloid Interface Sci. 1998;74:169-194. 8. Evan AP, Coe FL, Lingeman JE, et al. Mechanism of formation of human calcium oxalate renal stones on Randall’s plaque. Anat Rec. 2007;290:1315-1323. 9. Evan AP, Coe FL, Rittling SR, et al. Apatite plaque particles in inner medulla of kidneys of calcium oxalate stone formers: osteopontin localization. Kidney Int. 2005;68:145-154. 10. Hennis HL, Hennigar GR, Greene WB, et al. Intratubular calcium phosphate deposition in acute analgesic nephropathy in rabbits. Am J Pathol. 1982;106:356-363. 11. Johansson SL, Sakata T, Hasegawa R, et al. The effect of long-term administration of aspirin and sodium saccharin on the rat kidney. Toxicol Appl Pharmacol. 1986;86:80-92.
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12. Molland EA. Aspirin damage in the rat kidney in the intact animal and after unilateral nephrectomy. J Pathol. 1976;120:43-48. 13. Johansson S, Angervall L. Urothelial changes of the renal papillae in Sprague-Dawley rats induced by long term feeding of phenacetin. Acta Pathol Microbiol Scand. 1976;84:375-383. 14. Arnold L, Collins C, Starmer GA. Further studies of the acute effects of phenylbutazone, oxyphenbutazone and indomethacin on the rat kidney. Pathology. 1976;8:135-141. 15. Ellenbogen PH, Talner LB. Uroradiology of diabetes mellitus. Urology. 1976;8:413-419. 16. Di Tommaso L, Tolomelli B, Mezzini R, et al. Renal calcium phosphate and oxalate deposition in prolonged vitamin B6 deficiency: studies on a rat model of urolithiasis. BJU Int. 2002;89:571575. 17. Mandel NS, Henderson JD Jr, Hung LY, et al. A porcine model of calcium oxalate kidney stone disease. J Urol. 2004;171:1301-1303. 18. Stoller M, Low R, Shami G, et al. High resolution radiography if cadaveric kidneys: unraveling the mystery of Randall’s plaque formation. J Urol. 1996;156:1263-1266. 19. Gill WB, Jones KW, Ruggiero KJ. Protective effects of heparin and other sulfated glycosaminoglycans on crystal adhesion to injured urothelium. J Urol. 1982;127:152-154. 20. Lieske JC, Norris R, Swift H, et al. Adhesion, internalization and metabolism of calcium oxalate monohydrate crystals by renal epithelial cells. Kidney Int. 1997;52:1291-1301. 21. Steitz SA, Speer MY, McKee MD, et al. Osteopontin inhibits mineral deposition and promotes regression of ectopic calcification. Am J Pathol. 2002;161:2035-2046.
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22. Romberg RW, Werness PG, Riggs BL, et al. Inhibition of hydroxyapatite crystal growth by bone-specific and other calcium-binding proteins. Biochemistry. 1986;25:1176-1180. 23. Boskey AL, Maresca M, Ullrich W, et al. Osteopontin-hydroxyapatite interactions in vitro: inhibition of hydroxyapatite formation and growth in a gelatin-gel. Bone Miner. 1993;22:147-159. 24. Govindaraj A, Selvam R. An oxalate-binding protein with crystal growth promoter activity from human kidney stone matrix. BJU Int. 2002;90:336-344. 25. Yamate T, Kohri K, Umekawa T, et al. The effect of osteopontin on the adhesion of calcium oxalate crystals to Madin-Darby canine kidney cells. Eur Urol. 1996;30:388-393. 26. Lieske JC, Toback FG, Deganello S. Sialic acid-containing glycoproteins on renal cells determine nucleation of calcium oxalate dihydrate crystals. Kidney Int. 2001;60:1784-1791. 27. Lomashvili KA, Cobbs S, Hennigar RA, et al. Phosphate-induced vascular calcification: role of pyrophosphate and osteopontin. J Am Soc Nephrol. 2004;15:1392-1401. 28. Wilson JW, Werness PG, Smith LH. Inhibitors of crystal growth of hydroxyapatite: a constant composition approach. J Urol. 1985;134: 1255-1258. 29. Grases F, Ramis M, Costa-Bauzá A. Effects of phytate and pyrophosphate on brushite and hydroxyapatite crystallization: comparison with the action of other polyphosphates. Urol Res. 2000;28: 136-140. 30. Bhuskute NM, Yap WW, Wah TM. A retrospective evaluation of Randall’s plaque theory of nephrolithiasis with CT attenuation values. Eur J Radiol. 2009;72:470-472.
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