Reinjury risk of nano-calcium oxalate monohydrate and calcium oxalate dihydrate crystals on injured renal epithelial cells: aggravation of crystal adhesion and aggregation
Abstract:Background
Renal epithelial cell injury facilitates crystal adhesion to cell surface and serves as a key step in renal stone formation. However, the effects of cell injury on the adhesion of nano-calcium oxalate crystals and the nano-crystal-induced reinjury risk of injured cells remain unclear.
Methods
African green monkey renal epithelial (Vero) cells were injured with H
2
O
2
to establish a cell injury model. Cell… Show more
“…These data indicated that urine pH did not affect compositions of the proteins bound to the crystals. This strengthened the hypothesis mentioned above that the acidic pH caused cellular toxicity that finally led to membrane lipid asymmetry and finally enhancement of crystal-cell adhesion 36 , 37 . Figure 7 Effect of pH on COM-binding capacity of apical membrane proteins.…”
Section: Resultssupporting
confidence: 84%
“…Tissue and cellular injury is one of the potential factors aggravating kidney stone formation 36 , 37 . The injury can induce membrane lipid asymmetry and loss of cell polarity, as well as crystal-cell adhesion 36 , 37 . Our data showed that the cells underwent cytotoxicity at the highly acidic pH (Fig.…”
Urine pH has been thought to be an important factor that can modulate kidney stone formation. Nevertheless, there was no systematic evaluation of such pH effect. Our present study thus addressed effects of differential urine pH (4.0–8.0) on calcium oxalate (CaOx) crystallization, crystal-cell adhesion, crystal internalization into renal tubular cells, and binding of apical membrane proteins to the crystals. Microscopic examination revealed that CaOx monohydrate (COM), the pathogenic form, was crystallized with greatest size, number and total mass at pH 4.0 and least crystallized at pH 8.0, whereas COD was crystallized with the vice versa order. Fourier-transform infrared (FT-IR) spectroscopy confirmed such morphological study. Crystal-cell adhesion assay showed the greatest degree of crystal-cell adhesion at the most acidic pH and least at the most basic pH. Crystal internalization assay using fluorescein isothiocyanate (FITC)-labelled crystals and flow cytometry demonstrated that crystal internalization into renal tubular cells was maximal at the neutral pH (7.0). Finally, there were no significant differences in binding capacity of the crystals to apical membrane proteins at different pH. We concluded that the acidic urine pH may promote CaOx kidney stone formation, whereas the basic urine pH (i.e. by alkalinization) may help to prevent CaOx kidney stone disease.
“…These data indicated that urine pH did not affect compositions of the proteins bound to the crystals. This strengthened the hypothesis mentioned above that the acidic pH caused cellular toxicity that finally led to membrane lipid asymmetry and finally enhancement of crystal-cell adhesion 36 , 37 . Figure 7 Effect of pH on COM-binding capacity of apical membrane proteins.…”
Section: Resultssupporting
confidence: 84%
“…Tissue and cellular injury is one of the potential factors aggravating kidney stone formation 36 , 37 . The injury can induce membrane lipid asymmetry and loss of cell polarity, as well as crystal-cell adhesion 36 , 37 . Our data showed that the cells underwent cytotoxicity at the highly acidic pH (Fig.…”
Urine pH has been thought to be an important factor that can modulate kidney stone formation. Nevertheless, there was no systematic evaluation of such pH effect. Our present study thus addressed effects of differential urine pH (4.0–8.0) on calcium oxalate (CaOx) crystallization, crystal-cell adhesion, crystal internalization into renal tubular cells, and binding of apical membrane proteins to the crystals. Microscopic examination revealed that CaOx monohydrate (COM), the pathogenic form, was crystallized with greatest size, number and total mass at pH 4.0 and least crystallized at pH 8.0, whereas COD was crystallized with the vice versa order. Fourier-transform infrared (FT-IR) spectroscopy confirmed such morphological study. Crystal-cell adhesion assay showed the greatest degree of crystal-cell adhesion at the most acidic pH and least at the most basic pH. Crystal internalization assay using fluorescein isothiocyanate (FITC)-labelled crystals and flow cytometry demonstrated that crystal internalization into renal tubular cells was maximal at the neutral pH (7.0). Finally, there were no significant differences in binding capacity of the crystals to apical membrane proteins at different pH. We concluded that the acidic urine pH may promote CaOx kidney stone formation, whereas the basic urine pH (i.e. by alkalinization) may help to prevent CaOx kidney stone disease.
“…Using our method, in which the aggregation buffer was made to be saturated with calcium and oxalate ions before the seeded individual COM crystals were added, neocrystallization could be excluded and measurements of the amount and degree of crystal aggregates should be more precise. Also, our strategy of using this saturated aggregation buffer together with the fixed and controllable amount of the seeded COM crystals was that we did not want to have any effects from dissolution (such as when the crystals were seeded into other solutions, for example plain artificial urine or even deionized water, without saturation of calcium and oxalate ions) that can affect crystal sizes because sizes of COM crystals can affect crystal aggregation (Gan et al, 2016 ).…”
Crystal aggregation is one of the most crucial steps in kidney stone pathogenesis. However, previous studies of crystal aggregation were rarely done and quantitative analysis of aggregation degree was handicapped by a lack of the standard measurement. We thus performed an in vitro assay to generate aggregation of calcium oxalate monohydrate (COM) crystals with various concentrations (25–800 μg/ml) in saturated aggregation buffer. The crystal aggregates were analyzed by microscopic examination, UV-visible spectrophotometry, and GraphPad Prism6 software to define a total of 12 aggregation indices (including number of aggregates, aggregated mass index, optical density, aggregation coefficient, span, number of aggregates at plateau time-point, aggregated area index, aggregated diameter index, aggregated symmetry index, time constant, half-life, and rate constant). The data showed linear correlation between crystal concentration and almost all of these indices, except only for rate constant. Among these, number of aggregates provided the greatest regression coefficient (r = 0.997; p < 0.001), whereas the equally second rank included aggregated mass index and optical density (r = 0.993; p < 0.001 and r = −0.993; p < 0.001, respectively) and the equally forth were aggregation coefficient and span (r = 0.991; p < 0.001 for both). These five indices are thus recommended as the most appropriate indices for quantitative analysis of COM crystal aggregation in vitro.
“…Therefore, it is believed that fungal oligochitosan did not induce the formation of smaller crystals due to its small size. This is important because smaller crystals can be more toxic [ 69 , 70 ].…”
Section: Discussionmentioning
confidence: 99%
“…Some studies have shown that small-sized COM crystals are more toxic because they are more easily phagocytosed, and upon entering the cell, they cause an increase in oxidative stress leading to cell damage and death [ 69 , 70 ]. Thus, the results of this study show that fungal chitosan, unlike animal chitosan, does not favor the increase in the formation of CaOx crystals (mainly COM) and does not reduce the size of the crystals.…”
Animal chitosan (Chit-A) is gaining more acceptance in daily activities. It is used in a range of products from food supplements for weight loss to even raw materials for producing nanoparticles and hydrogel drug carriers; however, it has low antioxidant activity. Fungal oligochitosan (OChit-F) was identified as a potential substitute for Chit-A. Cunninghamella elegans is a fungus found in the Brazilian savanna (Caatinga) that produces OligoChit-F, which is a relatively poorly studied compound. In this study, 4 kDa OChit-F with a 76% deacetylation degree was extracted from C. elegans. OChit-F showed antioxidant activity similar to that of Chit-A in only one in vitro test (copper chelation) but exhibited higher activity than that of Chit-A in three other tests (reducing power, hydroxyl radical scavenging, and iron chelation). These results indicate that OChit-F is a better antioxidant than Chit-A. In addition, Chit-A significantly increased the formation of calcium oxalate crystals in vitro, particularly those of the monohydrate (COM) type; however, OChit-F had no effect on this process in vitro. In summary, OChit-F had higher antioxidant activity than Chit-A and did not induce the formation of CaOx crystals. Thus, OChit-F can be used as a Chit-A substitute in applications affected by oxidative stress.
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