Urolithiasis DOI 10.1007/s00240-015-0749-5
ORIGINAL PAPER
Aqueous extract of Costus arabicus inhibits calcium oxalate crystal growth and adhesion to renal epithelial cells Mitra R. de Cógáin · Michael P. Linnes · Hyo Jung Lee · Amy E. Krambeck · Julio Cezar de Mendonça Uchôa · Sung‑Hoon Kim · John C. Lieske
Received: 27 March 2014 / Accepted: 1 January 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Costus arabicus L. (C. arabicus) is a plant used in Brazilian folk medicine to treat urolithiasis; however, its mechanism of action is unclear. The interaction between calcium oxalate (CaOx) crystals and the renal epithelium is important in calculogenesis, and compounds that modulate this process represent candidate therapeutic agents for stone prevention. Therefore, we assessed the inhibitory activity of C. arabicus on CaOx crystallization and the interaction of CaOx crystals with the renal epithelium. A seeded CaOx monohydrate (COM) crystallization system was used to study the effect of C. arabicus on crystal growth. Madin Darby canine kidney (MDCK) cells were used to study [14C] COM crystal adhesion in the presence and absence of an aqueous extract of C. arabicus. Cytotoxicity was assessed using a tetrazolium (MTS) cell proliferation assay. Aqueous extracts of C. arabicus decreased crystal growth in a concentration-dependent fashion. Precoating crystals with C. arabicus extract prevented their adhesion to MDCK cells, while pretreating cells did not show any M. R. de Cógáin · A. E. Krambeck Department of Urology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA M. P. Linnes · S.‑H. Kim · J. C. Lieske (*) Division of Nephrology and Hypertension, Department of Internal Medicine, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA e-mail: [email protected] H. J. Lee · S.‑H. Kim (*) College of Oriental Medicine, Kyunghee University, 1 Hoegi‑dong, Dongdaemun‑gu, Seoul 131‑701, Republic of Korea e-mail: [email protected] J. C. de Mendonça Uchôa Department of Chemistry, Federal University of Alagoas, Maceio, Brazil
effect. The extract was non-cytotoxic in concentrations of at least 1 mg/ml, which is likely above concentrations achievable in the urine following oral ingestion and excretion. No inhibitory activity was found in hexane, methyl chloride, n-butanol and ethyl acetate fractions of an ethanol extract of the herb. An aqueous extract of C. arabicus may disrupt calculogenesis by interacting with CaOx crystal surfaces. Activity was present in the aqueous extract; therefore, this agent may be bioavailable when administered orally. Fractionation results suggest that the active agent might be a polar polysaccharide. Further identification and characterization along these lines may be warranted. Keywords Traditional medicine · Costus arabicus · Cell–crystal interaction · Crystallization · Herb · Nephrolithiasis
Introduction Urolithiasis is a highly prevalent condition in the United States, with up to 10 % of Americans diagnosed with urinary calculi during their lifetime, recurrence rates are up to 50 % [1]. Each episode can cause significant inconvenience, decreased productivity and wages, and in the most severe cases lead to renal dysfunction [2]. Medical therapies including thiazide diuretics, citrate compounds, and allopurinol appear to reduce stone recurrence rates, but can be poorly tolerated, may target only a single factor for stone formation, and appear to reduce repeat stone episodes by about half [3]. Therefore, additional preventative strategies would be welcomed. For centuries, various antiurolithic herbal therapies have been used, and in the Western world there is increasing interest in these compounds as possible agents for stone prevention and treatment [4]. A review of phytotherapeutic
13
Urolithiasis
agents for urolithiasis was performed by Gürocak and Küpeli in 2006 detailing the common mechanisms of action of various agents including diuretic properties, disaggregation of crystallization-promoting mucoproteins, alteration of cell–crystal interaction or perturbation of ionic compounds in urine [4]. Notably, the compounds identified act at different points in the crystallization cascade. Recently, renewed interest in complementary and adjunct stone therapies has arisen. One proposed mechanism of calculogenesis, the fixed particle theory, hypothesizes an interaction of crystals and the renal epithelium to mediate crystal retention and initiate stone formation [5]. The resulting tubular plugs appear to be composed of calcium phosphate, although prominent tubular plugs have also been observed in certain hyperoxaluric conditions, and injury or reaction to calcium oxalate crystals could have preceded [6, 7]. A second proposed mechanism of calculogenesis hypothesizes growth of calcium oxalate crystals on interstitial Randall’s plaque that has eroded through the urothelial surface [8, 9]. A third pathway described in the formation of stones is the precipitation of mineral in free space without attachment. Cystine stones might arise in this fashion [10]. Compounds that alter any of these processes could potentially inhibit stone formation and/or propagation. Costus arabicus L. (C. arabicus), also known as Costus spiralis var. hirsutus and commonly known as Cana agre, is a plant of the family Costaceae which has historically been used in Brazilian and other regional folk medicine to treat urinary symptoms and urolithiasis [11]. The whole plant is pulverized and dried, then used to brew therapeutic teas, which are thought to act via a diuretic as well other unknown physiologic mechanisms to expel urolithiasis and also potentially prevent stone growth. When a similar plant, C. spiralis, was administered to rats, it was found to decrease calcium oxalate (CaOx) crystallization upon zinc discs implanted in their bladders, suggesting that calculus formation may be impaired by this and similar herbs [11]. Additional evidence suggests these compounds have antimicrobial, antifungal, antioxidant, antiinflammatory, and immunomodulatory activities; however, its role in urinary stone formation has not been determined [12–17]. In the current study, we investigated the effect of C. arabicus on CaOx crystallization and the interaction of CaOx crystals with the renal epithelium.
as described by Pak et al. [18], with a specific activity of 300–450 cpm/μg. COM crystals were cuboidal to spindle shaped and uniformly small at 1–2 μm in largest diameter. Dulbecco–Vogt modified Eagle’s essential medium (DMEM), antibiotics and antimycotics were obtained from GIBCO (Grand Island, NY, USA) and fetal bovine serum (FBS) was provided by JRH (Lenexa, KS, USA). MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was obtained from Promega US (Madison, WI). Preparation of aqueous extract Dried C. arabicus was obtained from Dr. Julio Cezar de Mendonça Uchôa, Brasil, following visual authentication performed by taxonomist Rosbergue Lúcio da Silva at Herbário Mac-Instituto do Meio Ambiente do Estado de Alagoas (voucher specimen number 56833). An initial aqueous extract was produced from 10 g of the dried plant prepared in 250 ml of boiling distilled water for 10 min, with subsequent filtration through cheese cloth and large bore (11 μ) filter paper followed by lyophilization. The extract was prepared in this way to most closely mimic the way this herb is used in traditional medicine, as a therapeutic tea. The resulting powder was reconstituted in sterile water. Fractionation of the plant was performed at the College of Oriental Medicine at Kyunghee University. Dried C. arabicus (200 g) was extracted at room temperature using 80 % methanol for 24 h. After filtration, concentration, and freeze drying, a methanol extract was obtained (yield = 15.5 g). The remaining C. arabicus was reconstituted in 500 mL of distilled water and partitioned with an equal volume of n-hexane. The resulting aqueous phase was partitioned in succession with an equal volume of methyl chloride, ethyl acetate and n-butanol, respectively. The n-hexane, methyl chloride, ethyl acetate, and n-butanol fractions were concentrated and lyophilized. The percentage yields for the various fractions relative to the initial C. arabicus methanol extract were as follows: n-hexane fraction (2.8 g, 1.4 %), methyl chloride fraction (1.5 g, 0.75 %), ethyl acetate fraction (3 g, 1.5 %) and n-butanol fraction (3.3 g, 1.65 %). These fractions were then reconstituted in DMSO or distilled water to obtain appropriate final concentrations for our fractionation studies.
Methods
Cell culture
Reagents
Madin Darby canine kidney type I (MDCK-I) renal epithelial cells were donated by Carl Verkoelen (Erasmus University, Rotterdam, The Netherlands). Cells were plated in DMEM containing 10 % FBS and 1 % antibiotic/
[14C]-Radiolabeled calcium oxalate monohydrate (COM) crystals were prepared from supersaturated solutions,
13
Urolithiasis
antimycotic, and grown to confluence (1 × 106 cells/35 mm plate) in a humidified incubator at 37 °C. Cytotoxicity assay Cytotoxicity of the aqueous extract of C. arabicus was evaluated using an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] colorimetric assay twice, in triplicate (n = 6), with the mean result for each group used for comparison. 96-well microplates were seeded with the MDCK-I cells at a density of 1 × 104 cells per well, in 100 μl of DMEM with 10 % FBS, and incubated at 37 °C for 24 h. Cells were then treated in serum-free DMEM for 24 h, then with C. arabicus (0.01, 0.1, 1, 10, 100, 1,000 μg/ml) solubilized in DMEM for 24 h. MTS/PMS solution (20 μl; 2.0 ml MTS and 100 μl of PMS) was added to each well, cells were incubated for 1 h at 37 °C, and the optical density (490 nm) was measured. Cell viability was then calculated using the following equation:
Inhibition activity was calculated using the following formula: Inhibition activity = (C40 Average − CBl Average)/(C0 Average − C40 Average).
COM crystal binding assays
Cell viability (%) = [(OD (C. arabicus) − OD (Blank))/ (OD (Control) − OD (Blank))] × 100.
Confluent plates of MDCK-I cells (prepared as described above) were pretreated with C. arabicus extracts for 15 min or 24 h, three times, in triplicate (n = 9). The cells were then rinsed three times with phosphate-buffered saline (PBS, pH 7.4), and 50 μl of a [14C]COM crystal suspension was distributed across the cells. Two minutes later, the cells were rinsed three times with PBS and adherent crystals were then counted by scraping the lysed cell layer (using NaOH) into a scintillation vial and counting radioactivity. In order to assess the binding of crystals precoated with C. arabicus, [14C] COM crystals were prepared by adding extract concentrations and 100 μl of [14C] COM crystal slurry (8 μg/ml) to PBS-rinsed plates of confluent cells simultaneously. This procedure was performed four times, in triplicate (n = 12).
COM crystal growth assay
Statistical analysis
Sodium acetate buffer (NaCl 8.562 g, barbituric acid 0.6406 g, acetic acid 0.3 ml in 1 L sterile water, pH 5.7) was used to dilute C. arabicus extract fractions to 1, 10 and 100 μg/ml. 490 μl of calcium chloride solution (NaCl 7.745 g, CaCl2∙2H2O 0.294 g, barbituric acid 0.6406 g, acetic acid 0.3 ml in 1 L sterile water, pH 5.7) was added to 1.5 ml Eppendorf tubes. Then, extract concentrations (10 μl) were added to each tube (performed in duplicate), along with 500 μl of 14C-oxalic acid acetate buffer (1:100 dilution of glacial acetic acid 1.15 ml in 50 ml sterile water, 0.2736 g of Na oxalate into sodium acetate buffer and 250 μCi of 14C-oxalic acid with sodium acetate buffer). COM crystal slurry (100 μl; 1.8 mg/ml of COM crystals in sodium acetate buffer) was then added into each tube. Scintillation vials were filled with 5 ml of scintillation cocktail (Emulsifier Safe Scintillation mixture; PerkinElmer) and 100 μl of the mixture from each Eppendorf tube was transferred for assessment of radioactivity (this is initial count, C0). The remainder of the mixture in the Eppendorf tubes was allowed to crystallize for 40 min, then vortexed at 14,000 rpm × 5 min. Aliquots of the supernatant (100 μl) were pipetted into scintillation vials for assessment of radioactivity (this is final count, C40). Heparin (1,700–1,900 Da, 10.29 U/ml) was used as a positive control and sterile water was used as a blank (Bl). This procedure was performed in triplicate (n = 3) and the mean result for each group used for comparison.
All data were defined as a mean ± SD. Statistical significance between the C. arabicus groups and untreated controls was determined with the one-way analysis of variance using JMP 8.0 software, with a p
ORIGINAL PAPER
Aqueous extract of Costus arabicus inhibits calcium oxalate crystal growth and adhesion to renal epithelial cells Mitra R. de Cógáin · Michael P. Linnes · Hyo Jung Lee · Amy E. Krambeck · Julio Cezar de Mendonça Uchôa · Sung‑Hoon Kim · John C. Lieske
Received: 27 March 2014 / Accepted: 1 January 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Costus arabicus L. (C. arabicus) is a plant used in Brazilian folk medicine to treat urolithiasis; however, its mechanism of action is unclear. The interaction between calcium oxalate (CaOx) crystals and the renal epithelium is important in calculogenesis, and compounds that modulate this process represent candidate therapeutic agents for stone prevention. Therefore, we assessed the inhibitory activity of C. arabicus on CaOx crystallization and the interaction of CaOx crystals with the renal epithelium. A seeded CaOx monohydrate (COM) crystallization system was used to study the effect of C. arabicus on crystal growth. Madin Darby canine kidney (MDCK) cells were used to study [14C] COM crystal adhesion in the presence and absence of an aqueous extract of C. arabicus. Cytotoxicity was assessed using a tetrazolium (MTS) cell proliferation assay. Aqueous extracts of C. arabicus decreased crystal growth in a concentration-dependent fashion. Precoating crystals with C. arabicus extract prevented their adhesion to MDCK cells, while pretreating cells did not show any M. R. de Cógáin · A. E. Krambeck Department of Urology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA M. P. Linnes · S.‑H. Kim · J. C. Lieske (*) Division of Nephrology and Hypertension, Department of Internal Medicine, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA e-mail: [email protected] H. J. Lee · S.‑H. Kim (*) College of Oriental Medicine, Kyunghee University, 1 Hoegi‑dong, Dongdaemun‑gu, Seoul 131‑701, Republic of Korea e-mail: [email protected] J. C. de Mendonça Uchôa Department of Chemistry, Federal University of Alagoas, Maceio, Brazil
effect. The extract was non-cytotoxic in concentrations of at least 1 mg/ml, which is likely above concentrations achievable in the urine following oral ingestion and excretion. No inhibitory activity was found in hexane, methyl chloride, n-butanol and ethyl acetate fractions of an ethanol extract of the herb. An aqueous extract of C. arabicus may disrupt calculogenesis by interacting with CaOx crystal surfaces. Activity was present in the aqueous extract; therefore, this agent may be bioavailable when administered orally. Fractionation results suggest that the active agent might be a polar polysaccharide. Further identification and characterization along these lines may be warranted. Keywords Traditional medicine · Costus arabicus · Cell–crystal interaction · Crystallization · Herb · Nephrolithiasis
Introduction Urolithiasis is a highly prevalent condition in the United States, with up to 10 % of Americans diagnosed with urinary calculi during their lifetime, recurrence rates are up to 50 % [1]. Each episode can cause significant inconvenience, decreased productivity and wages, and in the most severe cases lead to renal dysfunction [2]. Medical therapies including thiazide diuretics, citrate compounds, and allopurinol appear to reduce stone recurrence rates, but can be poorly tolerated, may target only a single factor for stone formation, and appear to reduce repeat stone episodes by about half [3]. Therefore, additional preventative strategies would be welcomed. For centuries, various antiurolithic herbal therapies have been used, and in the Western world there is increasing interest in these compounds as possible agents for stone prevention and treatment [4]. A review of phytotherapeutic
13
Urolithiasis
agents for urolithiasis was performed by Gürocak and Küpeli in 2006 detailing the common mechanisms of action of various agents including diuretic properties, disaggregation of crystallization-promoting mucoproteins, alteration of cell–crystal interaction or perturbation of ionic compounds in urine [4]. Notably, the compounds identified act at different points in the crystallization cascade. Recently, renewed interest in complementary and adjunct stone therapies has arisen. One proposed mechanism of calculogenesis, the fixed particle theory, hypothesizes an interaction of crystals and the renal epithelium to mediate crystal retention and initiate stone formation [5]. The resulting tubular plugs appear to be composed of calcium phosphate, although prominent tubular plugs have also been observed in certain hyperoxaluric conditions, and injury or reaction to calcium oxalate crystals could have preceded [6, 7]. A second proposed mechanism of calculogenesis hypothesizes growth of calcium oxalate crystals on interstitial Randall’s plaque that has eroded through the urothelial surface [8, 9]. A third pathway described in the formation of stones is the precipitation of mineral in free space without attachment. Cystine stones might arise in this fashion [10]. Compounds that alter any of these processes could potentially inhibit stone formation and/or propagation. Costus arabicus L. (C. arabicus), also known as Costus spiralis var. hirsutus and commonly known as Cana agre, is a plant of the family Costaceae which has historically been used in Brazilian and other regional folk medicine to treat urinary symptoms and urolithiasis [11]. The whole plant is pulverized and dried, then used to brew therapeutic teas, which are thought to act via a diuretic as well other unknown physiologic mechanisms to expel urolithiasis and also potentially prevent stone growth. When a similar plant, C. spiralis, was administered to rats, it was found to decrease calcium oxalate (CaOx) crystallization upon zinc discs implanted in their bladders, suggesting that calculus formation may be impaired by this and similar herbs [11]. Additional evidence suggests these compounds have antimicrobial, antifungal, antioxidant, antiinflammatory, and immunomodulatory activities; however, its role in urinary stone formation has not been determined [12–17]. In the current study, we investigated the effect of C. arabicus on CaOx crystallization and the interaction of CaOx crystals with the renal epithelium.
as described by Pak et al. [18], with a specific activity of 300–450 cpm/μg. COM crystals were cuboidal to spindle shaped and uniformly small at 1–2 μm in largest diameter. Dulbecco–Vogt modified Eagle’s essential medium (DMEM), antibiotics and antimycotics were obtained from GIBCO (Grand Island, NY, USA) and fetal bovine serum (FBS) was provided by JRH (Lenexa, KS, USA). MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was obtained from Promega US (Madison, WI). Preparation of aqueous extract Dried C. arabicus was obtained from Dr. Julio Cezar de Mendonça Uchôa, Brasil, following visual authentication performed by taxonomist Rosbergue Lúcio da Silva at Herbário Mac-Instituto do Meio Ambiente do Estado de Alagoas (voucher specimen number 56833). An initial aqueous extract was produced from 10 g of the dried plant prepared in 250 ml of boiling distilled water for 10 min, with subsequent filtration through cheese cloth and large bore (11 μ) filter paper followed by lyophilization. The extract was prepared in this way to most closely mimic the way this herb is used in traditional medicine, as a therapeutic tea. The resulting powder was reconstituted in sterile water. Fractionation of the plant was performed at the College of Oriental Medicine at Kyunghee University. Dried C. arabicus (200 g) was extracted at room temperature using 80 % methanol for 24 h. After filtration, concentration, and freeze drying, a methanol extract was obtained (yield = 15.5 g). The remaining C. arabicus was reconstituted in 500 mL of distilled water and partitioned with an equal volume of n-hexane. The resulting aqueous phase was partitioned in succession with an equal volume of methyl chloride, ethyl acetate and n-butanol, respectively. The n-hexane, methyl chloride, ethyl acetate, and n-butanol fractions were concentrated and lyophilized. The percentage yields for the various fractions relative to the initial C. arabicus methanol extract were as follows: n-hexane fraction (2.8 g, 1.4 %), methyl chloride fraction (1.5 g, 0.75 %), ethyl acetate fraction (3 g, 1.5 %) and n-butanol fraction (3.3 g, 1.65 %). These fractions were then reconstituted in DMSO or distilled water to obtain appropriate final concentrations for our fractionation studies.
Methods
Cell culture
Reagents
Madin Darby canine kidney type I (MDCK-I) renal epithelial cells were donated by Carl Verkoelen (Erasmus University, Rotterdam, The Netherlands). Cells were plated in DMEM containing 10 % FBS and 1 % antibiotic/
[14C]-Radiolabeled calcium oxalate monohydrate (COM) crystals were prepared from supersaturated solutions,
13
Urolithiasis
antimycotic, and grown to confluence (1 × 106 cells/35 mm plate) in a humidified incubator at 37 °C. Cytotoxicity assay Cytotoxicity of the aqueous extract of C. arabicus was evaluated using an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] colorimetric assay twice, in triplicate (n = 6), with the mean result for each group used for comparison. 96-well microplates were seeded with the MDCK-I cells at a density of 1 × 104 cells per well, in 100 μl of DMEM with 10 % FBS, and incubated at 37 °C for 24 h. Cells were then treated in serum-free DMEM for 24 h, then with C. arabicus (0.01, 0.1, 1, 10, 100, 1,000 μg/ml) solubilized in DMEM for 24 h. MTS/PMS solution (20 μl; 2.0 ml MTS and 100 μl of PMS) was added to each well, cells were incubated for 1 h at 37 °C, and the optical density (490 nm) was measured. Cell viability was then calculated using the following equation:
Inhibition activity was calculated using the following formula: Inhibition activity = (C40 Average − CBl Average)/(C0 Average − C40 Average).
COM crystal binding assays
Cell viability (%) = [(OD (C. arabicus) − OD (Blank))/ (OD (Control) − OD (Blank))] × 100.
Confluent plates of MDCK-I cells (prepared as described above) were pretreated with C. arabicus extracts for 15 min or 24 h, three times, in triplicate (n = 9). The cells were then rinsed three times with phosphate-buffered saline (PBS, pH 7.4), and 50 μl of a [14C]COM crystal suspension was distributed across the cells. Two minutes later, the cells were rinsed three times with PBS and adherent crystals were then counted by scraping the lysed cell layer (using NaOH) into a scintillation vial and counting radioactivity. In order to assess the binding of crystals precoated with C. arabicus, [14C] COM crystals were prepared by adding extract concentrations and 100 μl of [14C] COM crystal slurry (8 μg/ml) to PBS-rinsed plates of confluent cells simultaneously. This procedure was performed four times, in triplicate (n = 12).
COM crystal growth assay
Statistical analysis
Sodium acetate buffer (NaCl 8.562 g, barbituric acid 0.6406 g, acetic acid 0.3 ml in 1 L sterile water, pH 5.7) was used to dilute C. arabicus extract fractions to 1, 10 and 100 μg/ml. 490 μl of calcium chloride solution (NaCl 7.745 g, CaCl2∙2H2O 0.294 g, barbituric acid 0.6406 g, acetic acid 0.3 ml in 1 L sterile water, pH 5.7) was added to 1.5 ml Eppendorf tubes. Then, extract concentrations (10 μl) were added to each tube (performed in duplicate), along with 500 μl of 14C-oxalic acid acetate buffer (1:100 dilution of glacial acetic acid 1.15 ml in 50 ml sterile water, 0.2736 g of Na oxalate into sodium acetate buffer and 250 μCi of 14C-oxalic acid with sodium acetate buffer). COM crystal slurry (100 μl; 1.8 mg/ml of COM crystals in sodium acetate buffer) was then added into each tube. Scintillation vials were filled with 5 ml of scintillation cocktail (Emulsifier Safe Scintillation mixture; PerkinElmer) and 100 μl of the mixture from each Eppendorf tube was transferred for assessment of radioactivity (this is initial count, C0). The remainder of the mixture in the Eppendorf tubes was allowed to crystallize for 40 min, then vortexed at 14,000 rpm × 5 min. Aliquots of the supernatant (100 μl) were pipetted into scintillation vials for assessment of radioactivity (this is final count, C40). Heparin (1,700–1,900 Da, 10.29 U/ml) was used as a positive control and sterile water was used as a blank (Bl). This procedure was performed in triplicate (n = 3) and the mean result for each group used for comparison.
All data were defined as a mean ± SD. Statistical significance between the C. arabicus groups and untreated controls was determined with the one-way analysis of variance using JMP 8.0 software, with a p
