Dual Modulatory Effects Of Diosmin On Calcium Oxalate Kidney Stone Formation Processes: Crystallization, Growth, Aggregation, Crystal-cell Adhesion, Internalization Into Renal Tubular Cells, And Invasion Through Extracellular Matrix
Mar 16, 2022
for more information:ali.ma@wecistanche.com
Supaporn Khamchuna,b,c,1, Sunisa Yoodeea,1, Visith Thongboonkerda,*
aMedical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
bDepartment of Medical Technology, School of Allied Health Sciences, University of Phayao, Phayao 56000, Thailand
cUnit of Excellence in Integrative Molecular Biomedicine, School of Allied Health Sciences, University of Phayao, Phayao 56000, Thailand
Keywords: Bioactive compound, Flavonoid, Inhibitor, Modulator, Nephrolithiasis, Promoter, Urolithiasis
ABSTRACT
Diosmin is a natural flavone glycoside (bioflavonoid) found in fruits and plants with several pharmacological activities. It has been widely used as a dietary supplement or therapeutic agent in various diseases/disorders. Although recommended, evidence of its protective mechanisms against kidney stone disease (nephrolithiasis/ urolithiasis), especially calcium oxalate (CaOx) monohydrate (COM) that is the most common type, remained unclear. In this study, we thus systematically evaluated the effects of diosmin (at 2.5–160 nM) on various stages of kidney stone formation processes, including COM crystallization, crystal growth, aggregation, crystal-cell adhesion, internalization into renal tubular cells and invasion through extracellular matrix (ECM). The results showed that diosmin had dose-dependent modulatory effects on all the mentioned COM kidney stone processes. Diosmin significantly increased COM crystal number and mass during crystallization, but reduced crystal size and growth. While diosmin promoted crystal aggregation, it inhibited crystal-cell adhesion and internalization into renal tubular cells. Finally, diosmin promoted crystal invasion through the ECM. Our data provide evidence demonstrating both inhibiting and promoting effects of diosmin on COM kidney stone formation processes. Based on these dual modulatory activities of diosmin, its anti-urolithiasis role is doubtful and cautions should be made for its use in kidney stone disease.
1. Introduction
Diosmin (3′,5,7-trihydroxy-4′-methoxyflavone-7-rhamnoglucoside) is a natural bioflavonoid commonly found in various plants and fruits, mainly Citrus spp. [1,2]. It can be synthesized or derived from the other flavonoid, hesperidin [1,2]. Diosmin alone or in combination with hesperidin is widely used as a phlebotropic drug for treatment of venous and lymphatic disorders such as chronic venous insufficiency and hemorrhoids [3,4]. In addition, diosmin exhibits several other biological and pharmacological activities, including antioxidant and anti-inflammatory activities [5,6], anti-mutagenic and anti-neoplastic properties [7,8], antibiotic effects [9], anti-hyperglycemic property [10], and protective effects against multi-organ damages, i.e.,
cardiovascular [11], retinal [12], kidney, liver and brain injury [13].
Kidney stone disease (or nephrolithiasis/urolithiasis) is caused by the solid calculi developed and deposited inside the kidney and affects humans in all areas of the globe with a high recurrence rate [14–17]. Among all various causative crystals, calcium oxalate (CaOx), especially monohydrate form (COM), is the most pathogenic and most common crystalline component found in the stone formers (patients with kidney stones) [18]. Mechanistically, COM crystals have the most potent adhesive capability and most cytotoxic effects on renal tubular epithelial cells [19,20]. During the stone pathogenesis, both Randall’s plaque model and intratubular hypothesis have common features of the COM stone formation processes, including COM crystallization, growth, aggregation, retention by surface adhesion on renal tubular cells or renal pelvis, internalization into the cells, and invasion into renal interstitium through extracellular matrix (ECM) [21,22].
Recently, many studies have focused on the drug discovery using medicinal plants/fruits and their bioactive compounds aiming for prevention of new and/or recurrent stone formation. A number of previous reports have shown that some bioactive compounds, especially phenolic compounds (polyphenol, flavonoid, flavone glycoside, etc.) extracted from several medicinal plants/fruits, have preventive effects against the pathogenic mechanisms of kidney stone disease both in vitro and in vivo [23–25]. Among these beneficial substances, a few reports have suggested that diosmin may prevent deposition of CaOx crystals inside kidney tissue in animal models [26,27]. However, its precise modulatory role (inhibition or promotion) and mechanisms (stages or stone formation processes) in COM kidney stone formation had not been investigated. The present study thus systematically evaluated the effects of diosmin (at 2.5–160 nM) on various stages of kidney stone formation processes, including crystallization, crystal growth, aggregation, crystal-cell adhesion, internalization into renal tubular cells and invasion through ECM.
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2. Materials and methods
2.1. Diosmin preparation
Diosmin (Tokyo Chemical Industry; Tokyo, Japan) was dissolved with 100% dimethyl sulfoxide (DMSO) (Sigma-Aldrich; St. Louis, MO) and working aliquots were made at the final concentrations of 2.5, 5, 10, 20, 40, 80 and 160 nM for investigating its effects on COM crystals. In addition, 100% DMSO was used as the negative control in all experiments.
2.2. Cell culture
MDCK renal tubular epithelial cell line derived from distal tubular segment of the nephron (ATCC; Manassas, VA) was propagated in Eagle’s minimum essential medium (MEM) (Gibco; Grand Island, NY) supplemented with 10% fetal bovine serum (FBS), 60 U/ml penicillin G (Sigma-Aldrich) and 60 μg/ml streptomycin (Sigma-Aldrich). The cells were maintained in a humidified incubator with 5% CO2 at 37 ◦C.
2.3. COM crystallization assay
COM crystallization was performed as described previously [28,29]. Briefly, 500 μl of 10 mM CaCl2⋅2H2O in crystallization buffer (containing 10 mM Tris-HCl and 90 mM NaCl) (pH 7.4) was added into each well of the 24-well plate (Corning Inc.; Corning, NY). An equal volume (4 μl) of crystallization buffer (blank control), DMSO (negative control) or diosmin (2.5, 5, 10, 20, 40, 80 or 160 nM) was mixed with CaCl2. Finally, 500 μl of 1.0 mM Na2C2O4 in the crystallization buffer was then added to make the final concentrations of CaCl2 and Na2C2O4 to 5 mM and 0.5 mM, respectively. The mixture was incubated at 25 ◦C for 1 h and then examined. Crystal images were captured randomly from at least 15 high-power fields (HPFs) under Nikon Eclipse Ti-S inverted phase-contrast light microscope (Nikon; Tokyo, Japan). Crystal size and number were measured from at least 15 HPFs for each sample using NIS Element D software version 4.11 (Nikon). Crystal mass was calculated from at least 100 crystals in 15 HPFs using the following formula: Crystal mass (µm2/HPF) = Average crystal size in each field (µm2) × Number of crystals in each field (/HPF) (1)
2.4. COM crystal growth assay
Crystal growth assay was performed as described previously [30,31].
Briefly, an equal volume (500 μl) of 10 mM CaCl2⋅2H2O and 1.0 mM Na2C2O4 in crystallization buffer was mixed (1:1) (v/v) in each well of the 24-well plate. The mixture was incubated at 25 ◦C for 1 h to allow complete crystallization. At this point (T0), an equal volume (4 μl) of crystallization buffer (blank control), DMSO (negative control) or diosmin (2.5, 5, 10, 20, 40, 80 or 160 nM) was added into each well and the mixture was further incubated for 60 min (T60). At T0 and T60, crystal images were captured randomly from at least 15 HPFs under Nikon Eclipse Ti-S inverted phase-contrast light microscope. Crystal sizes at T0 and T60 were measured using NIS Element D software version 4.11 (Nikon), whereas crystal growth (represented by Δ Crystal size) was calculated from at least 100 crystals in 15 HPFs using the following formula: Δ Crystal size (µm2) = Crystal size at T60 − Crystal size at T0 (2)
2.5. COM crystal aggregation assay
Crystal aggregation assay was performed as described previously [32,33]. COM crystals were generated as mentioned above in the crystal growth assay but with a larger volume in a 50-ml conical tube (Corning Inc.) and then harvested by centrifugation at 2000 g for 5 min. The supernatant was discarded, whereas COM crystals were washed three times with methanol. After another centrifugation at 2000 g for 5 min, methanol was discarded and the crystals were air-dried overnight at 25 ◦C. COM crystals (1000 µg dry weight) were resuspended in 1 ml of crystallization buffer in each well of the 6-well plate (Corning Inc.). An equal volume (4 μl) of crystallization buffer (blank control), DMSO (negative control) or diosmin (2.5, 5, 10, 20, 40, 80 or 160 nM) was added into the COM crystal suspension in each well. The plate was continuously shaken in a shaking incubator (Zhicheng; Shanghai, China) at 150 rpm and 25 ◦C for 1 h. Thereafter, formation of COM crystal aggregate (defined as “an assembly of three or more individual COM crystals that tightly joined together” [32]) was examined and imaged under Nikon Eclipse Ti-S inverted phase-contrast light microscope. Number of COM crystal aggregates was counted from at least 15 random HPFs per well.
2.6. COM crystal‑cell adhesion assay
Crystal-cell adhesion assay was performed as described previously [34,35]. Briefly, COM crystals were generated as mentioned above in a 50-ml conical tube and then harvested by centrifugation at 2000 g for 5 min. The supernatant was discarded, whereas COM crystals were washed three times with methanol. After another centrifugation at 2000 g for 5 min, methanol was discarded and the crystals were air-dried overnight at 25 ◦C. The crystals were decontaminated by UV light radiation for 30 min before intervention with the cells.
MDCK cells (at a density of 2 × 105 cells/well) were seeded and grown in each well of the 6-well plate (Corning Inc.) for 48 h to obtain confluent monolayer. The culture medium was then refreshed before adding COM crystals (100 µg dry weight crystals per ml culture medium). An equal volume (4 μl) of crystallization buffer (blank control), DMSO (negative control) or diosmin (2.5, 5, 10, 20, 40, 80 or 160 nM) was added into each well. The cells were further incubated in a humidified incubator with 5% CO2 at 37 ◦C for 1 h. Thereafter, the cells were vigorously washed with PBS five times and imaged under Nikon Eclipse Ti-S inverted phase-contrast light microscope. Number of the remaining COM crystals adhered onto renal tubular cell surface was counted from at least 15 random fields per well.
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2.7. COM crystal internalization assay
The fluorescence-labelled COM crystals were generated as described previously [36,37]. Compositions/concentrations of chemicals used for crystallization were the same as mentioned above for the plain (non-labelled) crystals, but 0.1 µg/ml fluorescein isothiocyanate (FITC) (Thermo Scientific Pierce; Rockford, IL) was added to CaCl2⋅2H2O solution prior to mixing with Na2C2O4. The subsequent steps (crystallization and harvesting) were performed as mentioned above, but in the dark.
To evaluate crystal internalization into renal tubular epithelial cells, MDCK cells (at a density of 2 ×105 cells/well) were seeded and grown in each well of the 6-well plate (Corning Inc.) for 48 h to obtain confluent monolayer. The cell monolayer was incubated with FITC-labelled COM crystals (1000 µg crystal/ml medium) in a humidified incubator with 5% CO2 at 37 ◦C for 1 h. Thereafter, the cells were washed with PBS and incubated with trypsin-EDTA solution to eliminate non-internalized (both adherent and non-adherent) crystals. Percentage of the cells with internalized crystals was quantified from a total of 10,000 acquired events using a flow cytometer (BD Accuri C6) (BD Biosciences; San Jose, CA). The cells incubated with plain (unlabelled) COM crystals were used for presetting the noise and threshold for the positive fluorescence signal. The cells with positive fluorescence signals were then counted and used for such percentage calculation.
2.8. COM crystal invasion through ECM assay
COM crystals were prepared as described above for crystal aggregation and crystal-cell adhesion assays. Crystal invasion assay was performed according to protocol established previously [38,39]. Briefly, a total of 20 µg COM crystals coated with crystallization buffer (blank control), DMSO (negative control), albumin (Sigma-Aldrich) (positive control) or diosmin (2.5, 5, 10, 20, 40, 80 or 160 nM) were added into 200 μl MEM. Subsequently, 200 μl of 0.3 pM Lys-plasminogen (Fitzgerald Industries international; Acton, MA) in PBS was mixed and incubated with the crystal-protein complex at 37 ◦C for 1 h. The unbound plasminogen was discarded by centrifugation at 2000 g for 5 min and the pellet was washed with PBS once. Thereafter, 100 μl of 0.15 pM urokinase plasminogen activator (uPA) (Fitzgerald Industries International) in PBS was mixed with the crystal-protein-plasminogen/plasmin complex. The mixture was then added on-top of the matrix gel inside the ECM migration chamber and incubated at 37 ◦C. After 24-h incubation, the solution remained on the upper part of the migration chamber was removed by using a gauze or tissue paper. The invaded COM crystals inside the matrix gel were then imaged using a light microscope with differential interference contrast (DIC) mode (Nikon H600L). The crystal invasion distance was measured and averaged from at least 15 low-power fields (LPFs) within the same chamber using NIS Element D software version 4.11 (Nikon).
2.9. Statistical analysis
All the above experiments were done in triplicate (three independent experiments) and the quantitative data are reported as mean ± SEM. Multiple comparisons were performed using one-way analysis of variance (ANOVA) with Tukey’s post-hoc test. Pearson correlation test was performed to determine the relationship between variables. All statistical analyses were done by using SPSS software (version 18) (IBM SPSS; Armonk, NY). P value less than 0.05 was considered statistically significant.
3. Results
3.1. Effect of diosmin on COM crystallization
Crystallization is one of the essential early steps of kidney stone formation processes of all types. After 1-h crystallization with or without diosmin at various concentrations (2.5, 5, 10, 20, 40, 80 or 160 nM), COM crystal size and number were measured and crystal mass was calculated. Crystallization buffer and the diluent DMSO served as the blank and negative controls, respectively. The data showed that all doses (2.5–160 nM) of disomin significantly reduced crystal size but increased crystal number in a dose-dependent manner when compared with blank and negative controls (Fig. 1A–1C). In concordance with the crystal number, crystal mass dose-dependently increased (Fig. 1D). Overall, these data indicate that diosmin promotes COM crystallization (neocrystals).
3.2. Effect of diosmin on COM crystal growth
The modulatory effect of diosmin on COM crystal growth was evaluated by measuring change in crystal size (Δ Crystal size) after 60-min further incubation following the completion of initial crystallization step, when neocrystals were absent. The results demonstrated that all doses (2.5–160nM) of disomin significantly reduced Δ Crystal size in a dose-dependent manner as compared with the blank and negative controls (Fig. 2). These findings indicate that diosmin inhibits COM crystal growth.
3.3. Effect of diosmin on COM crystal aggregation
In addition to crystal growth, aggregation of individual crystals that adhere tightly together is another important step during kidney stone pathogenesis. The high degree of crystal aggregation may ultimately lead to stone enlargement and obstruction of small renal tubular lumen. Comparing with blank and negative controls, diosmin at 10–160nM dose-dependently increased number of the COM crystal aggregates (Fig. 3). These results indicate that diosmin promotes COM crystal aggregation.
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3.4. Effect of diosmin on COM crystal‑cell adhesion
Retention of the causative crystals is one of the crucial steps for kidney stone formation and can be induced by crystal-cell adhesion that prevents the crystals from expelling together with the urine outflow. We thus evaluated the modulatory activity of diosmin on COM crystal-cell adhesion. The data revealed that diosmin at 5–160nM significantly reduced the adhesive capability of COM crystals on renal tubular cells in a dose-dependent manner as compared with blank and negative controls (Fig. 4). These data indicate that diosmin inhibits COM crystal-cell adhesion.
3.5. Effect of diosmin on COM crystal internalization into renal tubular cells
After adhesion, some COM crystals can be internalized into the renal tubular cells and cause several subsequent cellular responses [37,40]. Crystal internalization was evaluated by using FITC-labelled COM crystals and quantified by flow cytometry. Plain COM crystals (without labelling) was used to subtract technical noise and to ensure that the fluorescence intensity was from FITC signal. Comparing with blank and negative controls, percentage of the cells with internalized crystals was significantly decreased by diosmin at 10–160nM in a dose-dependent manner (Fig. 5). The findings indicate that disomin inhibits COM crystal internalization into renal tubular cells.
3.6. Effect of diosmin on COM crystal invasion through ECM
Crystal invasion through ECM is a pathogenic/destructive process during kidney stone pathogenesis that can trigger various inflammatory responses and cascades, thereby worsening the disease mechanisms. We examined this phenomenon by using an established protocol based on plasminogen-plasmin activity of the crystal-protein complex [38,39]. The results showed that all doses of disomin (2.5–160nM) significantly enhanced COM crystal invasion through the ECM in a dose-dependent manner (Fig. 6). Interestingly, diosmin at 160nM could promote the COM crystal invasion comparable to albumin, which is the known potent promoter for COM crystal invasion [41] (Fig. 6). These results indicate that diosmin strongly promotes COM crystal invasion through the ECM.
Fig. 1. Effect of diosmin on COM crystallization. Crystallization assay was performed with an equal volume (4μl) of crystallization buffer (blank control), DMSO (negative control) or diosmin (2.5–160nM). (A): Crystal morphology in each condition after 1-h crystallization. Original magnification was 400× for all panels. (B): Crystal size. (C): Crystal number. (D): Crystal mass (see Formula 1 in “Materials and Methods”) was analyzed from at least 100 crystals in 15 HPFs. Each bar represents mean ±SEM of the data derived from 3 independent experiments. * = p< 0.05 vs. blank control; # = p< 0.05 vs. DMSO.
Fig. 2. Effect of diosmin on COM crystal growth. Crystal growth assay was performed after the crystallization was complete (to prevent neocrystallization) with an equal volume (4μl) of crystallization buffer (blank control), DMSO (negative control) or diosmin (2.5–160nM). (A): Crystal morphology in each condition at T0 and T60. Original magnification was 400 × for all panels. (B)-(J): Histograms of crystal sizes measured from individual crystals at T0 and T60 in each group. (K): Δ Crystal size (see Formula 2 in “Materials and Methods”) was analyzed from at least 100 crystals in 15 HPFs. Each bar represents mean ± SEM of the data derived from 3 independent experiments. *= p < 0.05 vs. blank control; # = p < 0.05 vs. DMSO.
Fig. 3. Effect of diosmin on COM crystal aggregation. Crystal aggregation assay was performed with an equal volume (4μl) of crystallization buffer (blank control), DMSO (negative control) or diosmin (2.5–160nM). (A): Micrographs of the aggregated COM crystals (labelled with dotted circles). Original magnification was 400 ×for all panels. (B): Number of the crystal aggregates was counted from at least 15 random HPFs in each well. Each bar represents mean ± SEM of the data derived from 3 independent experiments. * = p< 0.05 vs. blank control; # =p < 0.05 vs. DMSO.
Fig. 4. Effect of diosmin on COM crystal-cell adhesion. Crystal‑cell adhesion assay was performed with an equal volume (4μl) of crystallization buffer (blank control), DMSO (negative control) or diosmin (2.5–160nM). (A): Micrographs of the remaining crystals that tightly adhered on the cell monolayer after removing the unbound crystals by vigorous washes with PBS. Original magnification was 200 × for all panels. (B): Number of the adhered crystals was counted from at least 15 random fields in each well. Each bar represents mean ±SEM of the data derived from 3 independent experiments. *= p <0.05 vs. blank control; # = p <0.05 vs. DMSO.
Fig. 5. Effect of diosmin on COM crystal internalization into renal tubular cells. Crystal internalization assay was performed using FITC-labelled COM crystals (FITC-COM), whereas the plain (non-labelled) COM crystals were used for background/noise subtraction. The assay was performed with an equal volume (4μl) of crystallization buffer (blank control), DMSO (negative control) or diosmin (2.5–160nM). (A): Flow cytometric dot-plot analysis of size (y-axis) and FITC-fluorescence intensity (x-axis) of the cells after removing non-internalized crystals with 0.1% trypsin/2.5mM EDTA. (B): Percentage of the cells with internalized FITC-labelled COM crystals. Each bar represents mean ± SEM of the data derived from 3 independent experiments. *= p <0.05 vs. FITC-COM + blank control; # = p< 0.05 vs. FITC-COM + DMSO.
Fig. 6. Effect of diosmin on COM crystal invasion through the ECM. Crystal invasion through ECM assay was performed using COM crystals coated with crystallization buffer (blank control), DMSO (negative control), albumin (positive control) or diosmin (2.5–160nM). (A): Micrographs of the COM crystals invaded or migrated through the ECM migration chamber. Original magnification was 100 × for all panels. (B): Crystal invasion distance was measured from at least 15 random LPFs in each chamber. Each bar represents mean ±SEM of the data derived from 3 independent experiments. *= p <0.05 vs. blank control; # =p < 0.05 vs. DMSO.
4. Discussion
Kidney stone disease is caused by formation of calculi inside the kidney and pelvocalyceal system. Common kidney stone formation processes include COM crystallization, growth, aggregation, retention by crystal-cell adhesion, and invasion through the renal interstitium rich with ECM [21,22]. There are several attempts to prevent this disease using a variety of medication and/or nutritional supplements. Many lines of recent evidence have reported that flavonoids, flavone glycosides and other compounds extracted from citrus fruits may have the preventive effects on kidney stone disease and other disorders [23–25]. Among these, diosmin, a natural flavone glycoside and hesperidin derivative found mainly in citrus fruits [1,2], has been suggested to play a preventive role against injury and damage of kidney tissue [13,42]. Moreover, its anti-urolithiasis role has been reported in a few previous studies using animal models [26,27]. Nevertheless, the beneficial effects and underlying mechanisms of diosmin in kidney stone prevention remained hazy. We thus examined the modulatory activity of diosmin on COM crystals. The systematic analyses were done on various stages of COM kidney stone formation, including crystallization, crystal growth, aggregation, crystal-cell adhesion, internalization into renal tubular cells and invasion through ECM.
After oral intake of diosmin, its plasma level in humans is ranged from 0.5 to 200ng/ml (or 0.8–300nM) [43,44]. The maximum plasma concentration (Cmax) is approximately 50ng/ml (or 85nM) [43,44]. Therefore, the dosages of diosmin used in our present study (2.5–160nM) were in the pharmacologically relevant range for its pharmacokinetics. Because DMSO was used as the diluent to completely dissolve diosmin per recommendation, DMSO was used as the negative control in this study in addition to the blank control to ensure that there were no effects from the diluent itself that might interfere with the data interpretation. In all assays, the data showed that there were no significant effects from DMSO observed and all the quantitative data derived from the negative control were comparable to those of the blank control.
COM crystallization assay revealed that all concentrations of diosmin (2.5, 5, 10, 20, 40, 80 and 160nM) could reduce COM crystal size but, on the other hand, increased the number of crystals. As crystal mass is the final product of both crystal size and number and is more relevant to reflect the degree of COM crystallization, we then evaluated whether diosmin affected this crystal index. The data showed that all concentrations of diosmin had the promoting effect on COM crystal mass, consistent with the data on crystal number. After crystallization, COM crystals can further grow to the larger size for next step of kidney stone formation.
In contrast to crystallization, diosmin at all concentrations exhibited the inhibitory effect on COM crystal growth in the dose-dependent manner. Some previous studies have reported that the solubility of CaOx crystals can be increased by derivatives of hydroxyanthraquinones with glycosylation [45]. Additionally, the presence of sugars in such bioactive compounds can bind with free calcium ion possibly due to the hydroxyl groups in the molecular structure, thereby inhibiting formation of CaOx crystals [45,46]. Diosmin might also affect the transition of the solubilized calcium and oxalate ions into the COM crystalline particles in renal tubular fluid in the process of COM crystallization based on this mechanism.
Nevertheless, COM crystal aggregation was promoted by 10–160nM diosmin, implicating the ability of diosmin to act as a linker or adhesive molecule for recruiting individual crystals to bind together to form the crystal aggregates. This binding also results in the progression of adhesive bridges among individual crystals and can induce the large structure formation of COM crystals, which easily contribute to the deposition of stone nidus inside the kidney [32].
COM crystal retention through the adhesion of crystals on renal tubular cell surfaces has been established as another important step for kidney stone formation [47]. Our data revealed that diosmin could reduce the adhesive capability of COM crystals to bind with apical surfaces of MDCK renal tubular cells. Some previous studies have shown that glycosaminoglycans (polysaccharide compounds) coated on CaOx crystals or expressed on renal tubular cells can interfere with the adhesive capability of CaOx crystals onto renal tubular cells [48]. Additionally, the expression of crystal receptors on renal tubular cells is one of the crucial mechanisms determining crystal-cell adhesion [21]. While CaOx crystals induces injury of apical membranes, particularly microvilli, some polyphenols such as epigallocatechin gallate (EGCG) can prevent the crystal-cell adhesion and cellular injury [49]. Moreover, flavonoid substance in various plant extracts has been previously reported to increase urinary pH level, leading to the inhibition of crystal-cell adhesion [50,51]. Perhaps, diosmin might also attenuate such cellular injury induced by COM, thereby reducing crystal-cell adhesion.
COM crystal internalization has been demonstrated to appear mainly by endocytosis via actin cytoskeleton-mediated macropinocytosis pathway [40]. Flavonoids such as quercetin have been reported to affect actin cytoskeleton required for macropinocytosis [52,53]. Our findings showed that diosmin significantly reduced the ability of MDCK cells to internalize COM crystals. Such inhibitory effect of diosmin might be associated with assembly or organization of actin cytoskeleton inside the cells that directly affects the macropinocytosis pathway [54,55].
After internalization, the COM crystals were degraded by endolysosomes resulting in increases of free calcium and oxalate ions in the renal interstitium [37]. Such increases in calcium and oxalate ions can lead to COM neocrystallization in the renal interstitium [56,57]. In addition, the presence of interstitial COM crystals may be from defects of tight junction and paracellular adhesion barriers, resulting in increased paracellular permeability and crystal translocation [58–60]. These crystals can then invade the renal interstitium through the ECM and subsequently trigger several inflammatory response and tissue damage [58–60]. In this study, we observed that the invasion of COM crystals through the ECM migration chamber was induced by all concentrations of diosmin. Diosmin might bind to the COM crystal surfaces and interacted with plasminogen-plasmin system that drove the crystal migration in the ECM migration chamber [38,39].
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Because diosmin induced both inhibiting and promoting effects on different various processes of COM stone formation, we therefore determined their relationship using Pearson correlation test. The correlation analysis revealed that crystal number inversely correlated with both crystal size and crystal growth (Δ Crystal size) (Fig. 7A and B). Crystal size strongly correlated with crystal growth (Fig. 7C) but inversely correlated with crystal mass (Fig. 7D). Finally, crystal mass strongly correlated with both crystal number and crystal aggregation (Fig. 7E and F). From the correlation analysis, the data indicate that crystal number and crystal mass are more relevant to reflect crystallization than crystal size (Fig. 7A, D and E). In addition, the size of neocrystals during crystallization are related to growth of the preformed crystals (Fig. 7C), whereas degree of crystallization (as reflected by crystal number and crystal mass) is closely related to degree of crystal aggregation (Fig. 7E and F). However, these correlations are most likely specific to the diosmin effects, whereas effects from other modulators may or may not have the same correlations. For example, fibronectin decreases crystal mass and inhibits crystal growth, but promotes crystal aggregation [31]. In addition, intact viable Escherichia coli increases crystal size and mass but has no effects on crystal number [33]. These data indicate that the correlations among various COM crystal assays are not universal for all modulators.
Finally, we have summarized the results obtained from various assays and integrate them with the pathogenic mechanisms of kidney stone disease (see schematics in Fig. 8). From the correlation analysis, diosmin promotes crystallization (Fig. 8A). Between crystallization and crystal growth, diosmin keeps a well balance by promoting crystallization but, on the other hand, inhibiting crystal growth (Fig. 8B). Concerning all the effects on COM crystals alone (without consideration of cells and ECM that also intervene), the promoting activity of diosmin is more prominent than its inhibiting activity on COM crystals as it promotes both crystallization and crystal aggregation, but inhibits only crystal growth (Fig. 8C). Although crystallization increases, the crystal size decreases and is associated with the growth inhibition (Fig. 7C). This data is consistent with the results reported by Kavanagh et al. [61–63], demonstrating that the increase of crystallization leads to the decline of supersaturation of calcium and oxalate ions in the solution, thereby decreasing the growth. However, crystal growth is not the only factor determining the kidney stone pathogenesis [64], particularly when the crystal mass overwhelms and is associated with the increase of crystal aggregation (Fig. 7F) that can increase a chance of crystal materials to get stuck in small tubular segments (in the intratubular hypothesis), thereby increasing the chance of stone formation. Moreover, a previous study has shown the data consistent with our findings, indicating that the increase in crystal number is associated with the increase of crystal aggregation and the stone enlargement [63].
Fig. 7. Correlations analysis. (A)–(F): Correlations among COM crystal number, crystal size, Δ Crystal size, crystal mass, and a number of crystal aggregates were analyzed by Pearson correlation test.
Fig. 8. Schematics summarizing the modulatory effects of diosmin on COM kidney stone formation processes. (A): Effects of diosmin on COM crystallization. (B): Effects of diosmin on COM crystallization and crystal growth. (C): Effects of diosmin on COM crystallization, growth and aggregation. (D): Effects of diosmin on the whole COM kidney stone formation processes.
When interactions with renal tubular cells and ECM are considered, the global picture of the dual effects of diosmin on COM kidney stone formation becomes much clearer (Fig. 8D). Diosmin inhibits crystal-cell adhesion, thereby reducing crystal internalization into the cells. This can be explained that the smaller size of COM crystals has less adhesive capability to binds the renal tubular cells compared with the larger ones as reported in our recent study [34] and previous study by another group [65]. The less adhesive capability of the smaller COM crystals is due to their less adhesive force to the cell surface and fewer numbers of the COM crystal receptors bound to them as determined by atomic force microscopy and proteome analysis, respectively [34]. Diosmin also inhibits crystal internalization. Note that the internalization process is a double-edged sword that needs careful interpretation. The internalization or endocytosis in one of the defense mechanisms that cells used for elimination of crystals by endolysosomes. However, degradation of the intact crystals generates free calcium and oxalate ions that can move from intracellular compartment to the renal interstitium, where they can generate neocrystals [56,57]. On the other hand, diosmin promotes crystal invasion through the ECM, which is one of the mechanisms important for kidney stone pathogenesis (particularly in the Randall’s plaque model) [58–60]. Overall, Fig. 8D summarizes all the dual modulatory effects of diosmin on COM kidney stone formation processes. It should be noted that the balance of these promoting and inhibiting effects cannot be precisely calculated (unlike a mathematic equation). Moreover, there are several other endogenous and exogenous factors that can also intervene the stone formation processes. Finally, diosmin also exhibits several indirect effects on kidney stone formation, including its antioxidant and anti-inflammatory properties [5,6] that should be taken into account. Therefore, the final outcome of these dual modulatory effects of diosmin on COM kidney stone formation needs further in vivo investigation and large-cohort prospective study.
5. Conclusions
In summary, we report herein the dual effects of diosmin on COM crystal modulation in a dose-dependent manner. While it inhibits COM crystal growth, crystal-cell adhesion, and internalization into renal tubular cells, diosmin promotes COM crystallization, aggregation, and invasion through the ECM. Therefore, its anti-urolithiasis role is doubtful and cautions should be made for its use in kidney stone disease.
CRediT authorship contribution statement
Supaporn Khamchun: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Data curation, Writing – original draft, Visualization, Sunisa Yoodee: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Data curation, Writing – original draft, Visualization, Visit Thongboonkerd: Conceptualization, Methodology, Software, Validation, Resources, Writing – review & editing, Supervision, Project administration, Funding acquisition.
Conflict of interest statement
The authors declare NO conflict of interest.
Acknowledgements
We are grateful for the technical assistance of Kittiya Suwannakud. This work was supported by The Office of National Higher Education Science Research and Innovation Policy Council (NXPO) through PMU-B and the Thailand Research Fund (IRN60W0004). VT is also supported by the “Chalermphrakiat” Grant, Faculty of Medicine Siriraj Hospital.
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