Urolithiasis (2015) 43 (Suppl 1):S33–S39 DOI 10.1007/s00240-014-0697-5
INVITED REVIEW
On the origin of calcium oxalate monohydrate papillary renal stones Fèlix Grases · Antonia Costa‑Bauzá · Carlo R. Bonarriba · Enrique C. Pieras · Rafael A. Fernández · Adrián Rodríguez
Received: 2 October 2013 / Accepted: 23 July 2014 / Published online: 3 August 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Calcium oxalate monohydrate (COM) papillary calculi can be initiated by subepithelial calcification of the renal papillae. Hydroxyapatite disruption of the papillary epithelial layer can become the nidus of a COM papillary calculus. This study evaluated the causes of papillary tissue calcifications in 60 patients with calcium oxalate lithiasis, 30 with COM papillary and 30 with calcium oxalate dihydrate (COD) calculi. Urinary redox potential was higher in the COM than the COD group, suggesting that the former is more deficient in antioxidants due to increased oxidative stress. Urinary calcium was significantly higher in the COD group, whereas urinary oxalate was significantly higher in the COM group, suggesting a greater degree of oxidative injury of renal cells. Evaluations of their diets showed that both groups consumed low amounts of phytate-rich products. Of chronic diseases possibly associated with urolithiasis, only the prevalence of gastroduodenal ulcer differed significantly, being higher in the COM group and suggesting that epithelial lesions are common to gastroduodenal ulcers and COM papillary renal stones. Occupational exposure to cytotoxic products occurred in 47 % of the COM and 27 % of the COD group, but this difference was not statistically significant. These findings indicate that oxidative stress is associated with injury to papillary tissue and that this is the origin of
F. Grases (*) · A. Costa‑Bauzá · R. A. Fernández · A. Rodríguez Laboratory of Renal Lithiasis Research, Faculty of Sciences, University Institute of Health Sciences Research (IUNICSIdISPa), University of Balearic Islands, 07122 Palma de Mallorca, Spain e-mail: [email protected] C. R. Bonarriba · E. C. Pieras Urology Service, University Hospital “Son Espases”, Palma de Mallorca, Spain
intrapapillary calcifications. The continuation of this process is due to modulators and/or deficiencies in inhibitors of crystallization. Identifying and eliminating the causes of injury may prevent recurrent episodes in patients with papillary COM calculi. Keywords Calcium oxalate · Papillary calcifications · Papillary renal calculi · Mechanism of formation
Introduction Renal stone formation results from a combination of various factors, some related to urine composition and others to kidney morphoanatomy. The components of stones (inorganic and organic) and their structures (macro and micro) can provide information about their mechanisms of formation and hence their etiology. Several studies to date have demonstrated connections between the structural features of calculi and etiological factors [1, 2]. Two types of calcium oxalate stones can occur, those composed of calcium oxalate dihydrate (COD) and calcium oxalate monohydrate (COM). COD crystals are thermodynamically unstable and can develop only under kinetically favorable conditions, including a higher degree of supersaturation (hypercalciuria and/or hyperoxaluria), deficits in crystallization inhibitors, and urodynamically appropriate conditions (e.g. urinary stagnation). Due to their thermodynamic instability, COD crystals slowly transform to stable COM crystals, mainly in contact with urine [3]. Nevertheless, the crystalline fine inner structure of calculi initially formed by COD crystals can be clearly identified by scanning electron microscopy [1]. COM calculi, in which COM crystals are directly formed from urine, can be classified into two types [2].
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COM papillary calculi, which account for about 13 % of urinary stones, are linked to papillary tissue, whereas COM calculi developed in renal cavities, which account for about 16 % of urinary stones, do not attach to the renal epithelium. The mechanism of development of these two types of calculi differs markedly, as do their fine microstructure. Studies of the mechanism of COM papillary formation began with the finding that a subepithelial calcification of renal papilla, resulting from the disruption of the papillary epithelial layer by a hydroxyapatite (HAP) plaque, becomes the nidus of a COM papillary calculus [4–9]. In patients susceptible to papillary calculi, the plaque is initiated in thin-loop basement membranes, in basement membranes of collecting tubules and in the vasa recta [6–9]. We have further assessed the causes of papillary tissue calcifications that initiate the formation of COM papillary calculi.
Patients and methods Patients This study included 60 patients with calcium oxalate renal lithiasis, 30 with COM papillary calculi and 30 with COD calculi; the two groups had the same sex and age distribution. Urine was obtained from each patient, and their case histories, lifestyles and dietary habits were determined by questionnaire. Each patient provided written informed consent, and the institutional review board of the Balearic Islands Community approved the study (No. IB1963/12PI). Renal calculi Spontaneously passed renal stones were dried, placed in sterile containers, and immediately studied. The outer and inner structures of the stones were analyzed by conventional stereoscopic microscopy (Optomic, Madrid, Spain), infrared spectrometry (Infrared Spectroscope Bruker IFS 66, Bruker, Ettlingen, Germany), and scanning electron microscopy (Hitachi S-3400N; Hitachi, Tokyo, Japan) coupled to X-ray microanalysis (XFlash Detector 4010, Bruker AXS, Berlin, Germany) [10]. A typical papillary COM stone (see Fig. 1a) consists of an eccentric core located near the concave region of the stone, near its site of attachment to the papillae, and a radially striated convex peripheral layer [2]. Scanning electron microscopy can detect microcomponents present in the core and can confirm the papillary origin of the calculus by examination of the concave external cavity. Thus, the presence of abundant organic matter and tubular apical cells demonstrates a point of attachment to the renal papillae. A typical unattached COM calculus (see Fig. 1b) consists of a symmetrically round stone with a central core,
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Urolithiasis (2015) 43 (Suppl 1):S33–S39
surrounded by columnar COM crystals emerging from the core, and by the absolute absence of a site of stone attachment to the epithelium [2]. A typical COD calculus consists primarily of randomly distributed pyramidal COD crystals, which form primary aggregates (see Fig. 1c). This morphology corresponds to calculi that remained in the kidney for a short time before being spontaneously passed. COD calculi remaining a long time in the kidney, in close contact with urine, have a disorganized morphology due to the transformation of COD to COM crystals; molds of COD crystals can be detected frequently (Fig. 1d). If this transformation is complete, the infrared spectra of these calculi are identical to those of directly formed COM stones. Analysis of serum and urine samples All subjects were on an unrestricted diet at the time of urine collection and none was receiving pharmacological treatment of any kind. Serum samples were obtained from the patients, and concentrations of creatinine, calcium, magnesium, phosphorus, and uric acid were measured as markers of renal function. Patients with renal failure or infected urine were excluded. Twenty-four-hour urine samples were collected 1–2 months after stone passage/removal in sterile flasks containing thymol as a preservative and immediately refrigerated. Total urine volume was recorded and the samples were stored at −20 °C until assayed. Calcium, magnesium and phosphorus concentrations were determined by inductively coupled plasma atomic spectroscopy. Uric acid and creatinine concentrations were measured using appropriate test kits (Roche Modular Analytics), and citrate and oxalate concentrations were determined using R-Biopharm enzymatic test kits. Two-hour urine samples were collected after overnight fasting and used to evaluate pH, phytate concentration and redox potential. The pH of each sample was measured with a glass electrode (Crison pH-meter) immediately after collection, thus avoiding changes in pH due to precipitation processes (calcium salts) that can occur during storage for 24 h. Urinary phytate analysis Two-hour urine samples were stored at 4 °C and transported to the laboratory in chilled containers. Each sample (20 mL) was acidified with HCl to pH 3, diluted with 20 mL of deionized water and quantitatively transferred to 100 mL beakers, each containing 0.5 g AG1-X8 resin without previous conditioning. Each mixture was stirred at 160 rpm for 15 min with an orbital stirrer to facilitate
Urolithiasis (2015) 43 (Suppl 1):S33–S39
S35
Fig. 1 Types of calcium oxalate renal calculi. a Section of a calcium oxalate monohydrate (COM) papillary stone. b Section of an unattached COM calculus. c Calcium oxalate dihydrate (COD) calculus
without transformation to COM. d COD calculus totally transformed to COM. Big COM crystals and moulds of COD can be observed
adsorption of phytate to the resin. The resin and urine were transferred to an empty 20 ml SPE tube with a frit and urine was passed through to separate it from the resin. The urine was discarded, and the resin was washed once with 120 ml 50 mM HCl and twice with 5 ml of deionized water while manually stirring with a glass rod. The resin was mixed with 1 ml of 2 M NaCl, while stirring at 180 rpm for 5 min. The eluate was collected, and the elution was repeated three times. The eluates (4 ml) were pooled and vortexed, and phytate was quantified colorimetrically [11]. Reagents (R1 and R2) were prepared daily, just before use. R1 consisted of a mixture of 1.2 ml of 4 mM Al(NO3)3·9H2O and 8.8 ml of 1.5 M acetic acid/acetate buffer, pH 5.2. R2 was a mixture of 1.2 ml of 4 mM xylenol orange and 8.8 ml H2O. Phytate standards, ranging in concentration from 2 to 12 μM, were prepared daily in 2 M NaCl. Assays were performed in 96-well plates after mixing 50 μL R1 and 250 μL of a standard or an eluate,
followed by the addition 30 s later of 50 μL of R2. The contents of each well were mixed and their absorbance at 550 nm was determined 45 min later using a microplate reader. Redox potential of urine samples The two-hour urine samples were immediately cooled to room temperature (25 °C), and their redox potential was measured using a Crison potentiometer, with a platinum electrode as the working electrode and a saturated calomel electrode as the reference electrode. Case history, lifestyle and dietary habits of patients All patients were interviewed about their family backgrounds of urolithiasis; their professional activities, especially those associated with possible exposure to cytotoxic compounds
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Urolithiasis (2015) 43 (Suppl 1):S33–S39
Table 1 Urinary pH, diuresis and excretion values of different urolithiasis-related biochemical parameters (values are expressed as mean ± SD) for calcium oxalate monohydrate papillary stone formers (papillary COM) and for calcium oxalate dihydrate stone formers (COD) COD
Diuresis (ml) pH Calcium (mmol/day) Magnesium (mmol/day)
1,257 ± 166 5.93 ± 0.49 4.76 ± 2.16 8.72 ± 10.68
1,306 ± 157 6.15 ± 0.76 7.52 ± 1.99* 7.51 ± 10.11
Phosphorus (mmol/day) Citrate (mmol/day) Creatinine (mmol/day) Oxalate (mmol/day) Uric acid (mmol/day)
30.47 ± 10.10 3.02 ± 1.49 12.27 ± 3.78 0.44 ± 0.16 4.22 ± 1.46
23.92 ± 11.71 2.78 ± 1.79 7.83 ± 0.93 0.22 ± 0.06* 2.96 ± 0.95
0.40 ± 0.20
0.38 ± 0.24
Phytate (μM) * p
INVITED REVIEW
On the origin of calcium oxalate monohydrate papillary renal stones Fèlix Grases · Antonia Costa‑Bauzá · Carlo R. Bonarriba · Enrique C. Pieras · Rafael A. Fernández · Adrián Rodríguez
Received: 2 October 2013 / Accepted: 23 July 2014 / Published online: 3 August 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Calcium oxalate monohydrate (COM) papillary calculi can be initiated by subepithelial calcification of the renal papillae. Hydroxyapatite disruption of the papillary epithelial layer can become the nidus of a COM papillary calculus. This study evaluated the causes of papillary tissue calcifications in 60 patients with calcium oxalate lithiasis, 30 with COM papillary and 30 with calcium oxalate dihydrate (COD) calculi. Urinary redox potential was higher in the COM than the COD group, suggesting that the former is more deficient in antioxidants due to increased oxidative stress. Urinary calcium was significantly higher in the COD group, whereas urinary oxalate was significantly higher in the COM group, suggesting a greater degree of oxidative injury of renal cells. Evaluations of their diets showed that both groups consumed low amounts of phytate-rich products. Of chronic diseases possibly associated with urolithiasis, only the prevalence of gastroduodenal ulcer differed significantly, being higher in the COM group and suggesting that epithelial lesions are common to gastroduodenal ulcers and COM papillary renal stones. Occupational exposure to cytotoxic products occurred in 47 % of the COM and 27 % of the COD group, but this difference was not statistically significant. These findings indicate that oxidative stress is associated with injury to papillary tissue and that this is the origin of
F. Grases (*) · A. Costa‑Bauzá · R. A. Fernández · A. Rodríguez Laboratory of Renal Lithiasis Research, Faculty of Sciences, University Institute of Health Sciences Research (IUNICSIdISPa), University of Balearic Islands, 07122 Palma de Mallorca, Spain e-mail: [email protected] C. R. Bonarriba · E. C. Pieras Urology Service, University Hospital “Son Espases”, Palma de Mallorca, Spain
intrapapillary calcifications. The continuation of this process is due to modulators and/or deficiencies in inhibitors of crystallization. Identifying and eliminating the causes of injury may prevent recurrent episodes in patients with papillary COM calculi. Keywords Calcium oxalate · Papillary calcifications · Papillary renal calculi · Mechanism of formation
Introduction Renal stone formation results from a combination of various factors, some related to urine composition and others to kidney morphoanatomy. The components of stones (inorganic and organic) and their structures (macro and micro) can provide information about their mechanisms of formation and hence their etiology. Several studies to date have demonstrated connections between the structural features of calculi and etiological factors [1, 2]. Two types of calcium oxalate stones can occur, those composed of calcium oxalate dihydrate (COD) and calcium oxalate monohydrate (COM). COD crystals are thermodynamically unstable and can develop only under kinetically favorable conditions, including a higher degree of supersaturation (hypercalciuria and/or hyperoxaluria), deficits in crystallization inhibitors, and urodynamically appropriate conditions (e.g. urinary stagnation). Due to their thermodynamic instability, COD crystals slowly transform to stable COM crystals, mainly in contact with urine [3]. Nevertheless, the crystalline fine inner structure of calculi initially formed by COD crystals can be clearly identified by scanning electron microscopy [1]. COM calculi, in which COM crystals are directly formed from urine, can be classified into two types [2].
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COM papillary calculi, which account for about 13 % of urinary stones, are linked to papillary tissue, whereas COM calculi developed in renal cavities, which account for about 16 % of urinary stones, do not attach to the renal epithelium. The mechanism of development of these two types of calculi differs markedly, as do their fine microstructure. Studies of the mechanism of COM papillary formation began with the finding that a subepithelial calcification of renal papilla, resulting from the disruption of the papillary epithelial layer by a hydroxyapatite (HAP) plaque, becomes the nidus of a COM papillary calculus [4–9]. In patients susceptible to papillary calculi, the plaque is initiated in thin-loop basement membranes, in basement membranes of collecting tubules and in the vasa recta [6–9]. We have further assessed the causes of papillary tissue calcifications that initiate the formation of COM papillary calculi.
Patients and methods Patients This study included 60 patients with calcium oxalate renal lithiasis, 30 with COM papillary calculi and 30 with COD calculi; the two groups had the same sex and age distribution. Urine was obtained from each patient, and their case histories, lifestyles and dietary habits were determined by questionnaire. Each patient provided written informed consent, and the institutional review board of the Balearic Islands Community approved the study (No. IB1963/12PI). Renal calculi Spontaneously passed renal stones were dried, placed in sterile containers, and immediately studied. The outer and inner structures of the stones were analyzed by conventional stereoscopic microscopy (Optomic, Madrid, Spain), infrared spectrometry (Infrared Spectroscope Bruker IFS 66, Bruker, Ettlingen, Germany), and scanning electron microscopy (Hitachi S-3400N; Hitachi, Tokyo, Japan) coupled to X-ray microanalysis (XFlash Detector 4010, Bruker AXS, Berlin, Germany) [10]. A typical papillary COM stone (see Fig. 1a) consists of an eccentric core located near the concave region of the stone, near its site of attachment to the papillae, and a radially striated convex peripheral layer [2]. Scanning electron microscopy can detect microcomponents present in the core and can confirm the papillary origin of the calculus by examination of the concave external cavity. Thus, the presence of abundant organic matter and tubular apical cells demonstrates a point of attachment to the renal papillae. A typical unattached COM calculus (see Fig. 1b) consists of a symmetrically round stone with a central core,
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Urolithiasis (2015) 43 (Suppl 1):S33–S39
surrounded by columnar COM crystals emerging from the core, and by the absolute absence of a site of stone attachment to the epithelium [2]. A typical COD calculus consists primarily of randomly distributed pyramidal COD crystals, which form primary aggregates (see Fig. 1c). This morphology corresponds to calculi that remained in the kidney for a short time before being spontaneously passed. COD calculi remaining a long time in the kidney, in close contact with urine, have a disorganized morphology due to the transformation of COD to COM crystals; molds of COD crystals can be detected frequently (Fig. 1d). If this transformation is complete, the infrared spectra of these calculi are identical to those of directly formed COM stones. Analysis of serum and urine samples All subjects were on an unrestricted diet at the time of urine collection and none was receiving pharmacological treatment of any kind. Serum samples were obtained from the patients, and concentrations of creatinine, calcium, magnesium, phosphorus, and uric acid were measured as markers of renal function. Patients with renal failure or infected urine were excluded. Twenty-four-hour urine samples were collected 1–2 months after stone passage/removal in sterile flasks containing thymol as a preservative and immediately refrigerated. Total urine volume was recorded and the samples were stored at −20 °C until assayed. Calcium, magnesium and phosphorus concentrations were determined by inductively coupled plasma atomic spectroscopy. Uric acid and creatinine concentrations were measured using appropriate test kits (Roche Modular Analytics), and citrate and oxalate concentrations were determined using R-Biopharm enzymatic test kits. Two-hour urine samples were collected after overnight fasting and used to evaluate pH, phytate concentration and redox potential. The pH of each sample was measured with a glass electrode (Crison pH-meter) immediately after collection, thus avoiding changes in pH due to precipitation processes (calcium salts) that can occur during storage for 24 h. Urinary phytate analysis Two-hour urine samples were stored at 4 °C and transported to the laboratory in chilled containers. Each sample (20 mL) was acidified with HCl to pH 3, diluted with 20 mL of deionized water and quantitatively transferred to 100 mL beakers, each containing 0.5 g AG1-X8 resin without previous conditioning. Each mixture was stirred at 160 rpm for 15 min with an orbital stirrer to facilitate
Urolithiasis (2015) 43 (Suppl 1):S33–S39
S35
Fig. 1 Types of calcium oxalate renal calculi. a Section of a calcium oxalate monohydrate (COM) papillary stone. b Section of an unattached COM calculus. c Calcium oxalate dihydrate (COD) calculus
without transformation to COM. d COD calculus totally transformed to COM. Big COM crystals and moulds of COD can be observed
adsorption of phytate to the resin. The resin and urine were transferred to an empty 20 ml SPE tube with a frit and urine was passed through to separate it from the resin. The urine was discarded, and the resin was washed once with 120 ml 50 mM HCl and twice with 5 ml of deionized water while manually stirring with a glass rod. The resin was mixed with 1 ml of 2 M NaCl, while stirring at 180 rpm for 5 min. The eluate was collected, and the elution was repeated three times. The eluates (4 ml) were pooled and vortexed, and phytate was quantified colorimetrically [11]. Reagents (R1 and R2) were prepared daily, just before use. R1 consisted of a mixture of 1.2 ml of 4 mM Al(NO3)3·9H2O and 8.8 ml of 1.5 M acetic acid/acetate buffer, pH 5.2. R2 was a mixture of 1.2 ml of 4 mM xylenol orange and 8.8 ml H2O. Phytate standards, ranging in concentration from 2 to 12 μM, were prepared daily in 2 M NaCl. Assays were performed in 96-well plates after mixing 50 μL R1 and 250 μL of a standard or an eluate,
followed by the addition 30 s later of 50 μL of R2. The contents of each well were mixed and their absorbance at 550 nm was determined 45 min later using a microplate reader. Redox potential of urine samples The two-hour urine samples were immediately cooled to room temperature (25 °C), and their redox potential was measured using a Crison potentiometer, with a platinum electrode as the working electrode and a saturated calomel electrode as the reference electrode. Case history, lifestyle and dietary habits of patients All patients were interviewed about their family backgrounds of urolithiasis; their professional activities, especially those associated with possible exposure to cytotoxic compounds
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Urolithiasis (2015) 43 (Suppl 1):S33–S39
Table 1 Urinary pH, diuresis and excretion values of different urolithiasis-related biochemical parameters (values are expressed as mean ± SD) for calcium oxalate monohydrate papillary stone formers (papillary COM) and for calcium oxalate dihydrate stone formers (COD) COD
Diuresis (ml) pH Calcium (mmol/day) Magnesium (mmol/day)
1,257 ± 166 5.93 ± 0.49 4.76 ± 2.16 8.72 ± 10.68
1,306 ± 157 6.15 ± 0.76 7.52 ± 1.99* 7.51 ± 10.11
Phosphorus (mmol/day) Citrate (mmol/day) Creatinine (mmol/day) Oxalate (mmol/day) Uric acid (mmol/day)
30.47 ± 10.10 3.02 ± 1.49 12.27 ± 3.78 0.44 ± 0.16 4.22 ± 1.46
23.92 ± 11.71 2.78 ± 1.79 7.83 ± 0.93 0.22 ± 0.06* 2.96 ± 0.95
0.40 ± 0.20
0.38 ± 0.24
Phytate (μM) * p
