Study on Fu-Fang-Jin-Qian-Cao Inhibiting Autophagy in Calcium Oxalate-induced Renal Injury by UHPLC/Q-TOF-MS-based Metabonomics and Network Pharmacology Approaches.

INTRODUCTION: Fu-Fang-Jin-Qian-Cao is a Chinese herbal preparation used to treat urinary calculi. Fu-Fang-Jin-Qian-Cao can protect renal tubular epithelial cells from calcium oxalateinduced renal injury by inhibiting ROS-mediated autopathy. The mechanism still needs further exploration. Metabonomics is a new subject; the combination of metabolomics and network pharmacology can find pathways for drugs to act on targets more efficiently. METHODS: Comprehensive metabolomics and network pharmacology to study the mechanism of Fu-Fang-Jin-Qian-Cao inhibiting autophagy in calcium oxalate-induced renal injury. Based on UHPLC-Q-TOF-MS, combined with biochemical analysis, a mice model of Calcium oxalateinduced renal injury was established to study the therapeutic effect of Fu-Fang-Jin-Qian-Cao. Based on the network pharmacology, the target signaling pathway and the protective effect of Fu- Fang-Jin-Qian-Cao on Calcium oxalate-induced renal injury by inhibiting autophagy were explored. Autophagy-related proteins LC3-II, BECN1, ATG5, and ATG7 were studied by immunohistochemistry. RESULTS: Combining network pharmacology and metabolomics, 50 differential metabolites and 2482 targets related to these metabolites were found. Subsequently, the targets enriched in PI3KAkt, MAPK and Ras signaling pathways. LC3-II, BECN1, ATG5 and ATG7 were up-regulated in Calcium oxalate-induced renal injury. All of them could be reversed after the Fu-Fang-Jin-Qian- Cao treatment. CONCLUSIONS: Fu-Fang-Jin-Qian-Cao can reverse ROS-induced activation of the MAPK signaling pathway and inhibition of the PI3K-Akt signaling pathway, thereby reducing autophagy damage of renal tubular epithelial cells in Calcium oxalate-induced renal injury.

Preprotection of Tea Polysaccharides with Different Molecular Weights Can Reduce the Adhesion between Renal Epithelial Cells and Nano-Calcium Oxalate Crystals.

Crystal adhesion is an important link in the formation of kidney stones. This study investigated and compared the adhesion differences between nano-calcium oxalate monohydrate (COM) and human renal proximal tubule epithelial (HK-2) cells before and after treatment with tea polysaccharides (TPSs) TPS0, TPS1, TPS2, and TPS3 with molecular weights of 10.88, 8.16, 4.82, and 2.31 kDa, respectively. TPS treatment effectively reduced the damage of COM to HK-2 cells, thereby resulting in increased cell activity, decreased release of lactate dehydrogenase, cell morphology recovery, decreased level of reactive oxygen species, increased mitochondrial membrane potential, increased lysosomal integrity, decreased expression of adhesion molecule osteopontin and eversion of phosphatidylserine, and decreased crystal adhesion. Among the TPSs, TPS2 with moderate molecular weight had the best protective effect on cells and the strongest effect on the inhibition of crystal adhesion. Thus, TPS2 may be a potential anticalculus drug.

Comparison of the adhesion of calcium oxalate monohydrate to HK-2 cells before and after repair using tea polysaccharides.

Background: Kidney stone formation is closely related to renal epithelial cell damage and the adhesion of calcium oxalate crystals to cells. Methods: In this research, the adhesion of human kidney proximal tubular epithelial cells (HK-2) to calcium oxalate monohydrate crystals with a size of approximately 100 nm was studied. In addition, the inhibition of crystal adhesion by four tea polysaccharides (TPS0, TPS1, TPS2, and TPS3) with the molecular weights of 10.88, 8.16, 4.82, and 2.31 kDa, respectively were compared. Results: When oxalic acid-damaged HK-2 cells were repaired, cell viability increased. By contrast, reactive oxygen species level, phosphatidylserine eversion, and osteopontin expression decreased, thus indicating that tea polysaccharides have a repairing effect on damaged HK-2 cells. Moreover, after repairing the damaged cells, the amount of adherent crystals was reduced. The repair effect of tea polysaccharides is closely related to molecular weight, and TPS2 with the moderate molecular weight displayed the best repair effect. Conclusion: These results suggest that tea polysaccharides, especially TPS2, may inhibit the formation and recurrence of calcium oxalate kidney stones.

Calcium oxalate druses affect leaf optical properties in selenium-treated Fagopyrum tataricum.

Plants of the genus Fagopyrum contain high levels of crystalline calcium oxalate (CaOx) deposits, or druses, that can affect the leaf optical properties. As selenium has been shown to modify the uptake and accumulation of metabolically important elements such as calcium, we hypothesised that the numbers of druses can be altered by selenium treatment, and this would affect the leaf optical properties. Tartary buckwheat (Fagopyrum tataricum Gaertn.) was grown outdoors in an experimental field. At the beginning of flowering, plants were foliarly sprayed with sodium selenate solution at 10 mg selenium L-1 or only with water. Plant morphological, biochemical, physiological and optical properties were examined, along with leaf elemental composition and content. Se spraying did not affect leaf biochemical and functional properties. However, it increased leaf thickness and the contents of Se in the leaves, and decreased the density of calcium oxalate druses in the leaves. Except Se content, Se spraying did not affect contents of other elements in leaves, including total calcium per dry mass of leaf tissue. Redundancy analysis showed that of all parameters tested, only the calcium oxalate druses parameters were significant in explaining the variability of the leaf reflectance and transmittance spectra. The density of CaOx druses positively correlated with the reflectance in the blue, green, yellow and UV-B regions of the spectrum, while the area of CaOx druses per mm2 of leaf transection area positively correlated with the transmittance in the green and yellow regions of the spectrum.

Response of renal tubular cells to differential types and doses of calcium oxalate crystals: Integrative proteome network analysis and functional investigations.

We have previously identified changes in the cellular proteome of renal tubular cells induced by low-dose (100 µg/mL) and high-dose (1000 µg/mL) calcium oxalate monohydrate (COM) and dihydrate (COD) crystals. However, the functional significance of such expression data remained unclear. In this study, we performed comparative analyses and functional investigations of four proteomic datasets to define potential mechanisms by which renal tubular cells responded to differential crystal types and doses. The data showed that high-dose induced greater changes than low-dose, whereas COM induced more changes than COD. Luciferin-luciferase ATP assay revealed increased intracellular ATP level by high-dose of both COM and COD. OxyBlot assay and Western blotting showed accumulated intracellular oxidized proteins but decreased ubiquitinated proteins by high-dose of both crystals. Flow cytometric analysis of cell death showed that high-dose of both crystals, particularly COM, significantly increased cell death. Also, crystal adhesion assay showed higher degree of cell-crystal adhesion in high-dose and COM when compared to low-dose and COD, respectively. Finally, pretreatment of epigallocatechin-3-gallate revealed a protective effect on COM/COD crystals-induced oxidative stress and cell-crystal adhesion. Collectively, these data may provide a better understanding of cellular responses of renal tubular cells to COM/COD crystals in kidney stone disease.

Urinary metabonomics elucidate the therapeutic mechanism of Orthosiphon stamineus in mouse crystal-induced kidney injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Orthosiphon stamineus (OS), a traditional Chinese herb, is often used for promoting urination and treating nephrolithiasis. AIM OF THE STUDY: Urolithiasis is a major worldwide public health burden due to its high incidence of recurrence and damage to renal function. However, the etiology for urolithiasis is not well understood. Metabonomics, the systematic study of small molecule metabolites present in biological samples, has become a valid and powerful tool for understanding disease phenotypes. In this study, a urinary metabolic profiling analysis was performed in a mouse model of renal calcium oxalate crystal deposition to identify potential biomarkers for crystal-induced renal damage and the anti-crystal mechanism of OS. MATERIALS AND METHODS: Thirty six mice were randomly divided into six groups including Saline, Crystal, Cystone and OS at dosages of 0.5g/kg, 1g/kg, and 2g/kg. A metabonomics approach using ultra-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) was developed to perform the urinary metabolic profiling analysis. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were utilized to identify differences between the metabolic profiles of mice in the saline control group and crystal group. RESULTS: Using partial least squares-discriminant analysis, 30 metabolites were identified as potential biomarkers of crystal-induced renal damage. Most of them were primarily involved in amino acid metabolism, taurine and hypotaurine metabolism, purine metabolism, and the citrate cycle (TCA). After the treatment with OS, the levels of 20 biomarkers had returned to the levels of the control samples. CONCLUSIONS: Our results suggest that OS has a protective effect for mice with crystal-induced kidney injury via the regulation of multiple metabolic pathways primarily involving amino acid, energy and choline metabolism.

Aqueous extract of Costus arabicus inhibits calcium oxalate crystal growth and adhesion to renal epithelial cells.

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 [(14)C] 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 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.

Preventive effect of specific antioxidant on oxidative renal cell injury associated with renal crystal formation.

OBJECTIVE: To investigate whether calcium oxalate monohydrate (COM), a key element of hyperoxaluria, would induce renal cell injury through oxidative stress and also whether certain antioxidants could prevent chemically induced renal crystal formation in rats. MATERIALS AND METHODS: COM-exerted oxidative stress on the kidney epithelial Madin-Darby canine kidney cells was assessed using the lipid peroxidation assay. Glyoxalase I (Gly-I) activity was also determined. Two antioxidants, vitamin C and N-acetylcysteine (NAC), were then tested to determine whether they could abolish such oxidative stress in Madin-Darby canine kidney cells. Both antioxidants were also tested to determine whether they might prevent or reduce renal crystal formation induced with ethylene glycol (EG) and vitamin D3 (VD3) in Wistar rats. RESULTS: COM (200 µg/mL) demonstrated ∼1.3-fold greater oxidative stress with a significant reduction in cell viability and Gly-I activity compared with controls. However, such adverse events were almost completely prevented with NAC but not with vitamin C. In the animal study, no renal crystals were seen in the sham group. However, numerous crystals, with reduced Gly-I activity and elevated oxidative stress, were found in the EG-VD3 group. However, markedly (>70%) fewer crystals, with full Gly-I activity and diminished oxidative stress, were detected in the EG-VD3+NAC group. CONCLUSION: COM exerted oxidative stress on Madin-Darby canine kidney cells, leading to cell viability reduction and Gly-I inactivation, with NAC fully preventing such adverse consequences. Similarly, numerous crystals with Gly-I inactivation and elevated oxidative stress seen in the rats (EG-VD3) were also significantly prevented with NAC supplement. Thus, NAC might have clinical implications in preventing oxidative renal cell injury and, ultimately, kidney stone formation.

[Study on irritation of calcium oxalate crystal in Araceae plants].

OBJECTIVE: To validate the irritation effects of calcium oxalate crystal in several herbal drugs which come from Araceae plants. METHOD: Compared the irritation of pure calcium oxalate crystals isolated from the raw rhizome of Typhonium flagelliforme, T. giganteum and Arisaema erubescens and studied the quantity and irritating effect relationship of different concentration suspensions of needle-like calcium oxalate crystals by using the model of rabbits' eyes. RESULT: Calcium oxalate crystals isolated from above three rau rhizome typhonium rhizome showed strong irritation effects on rabbits' eyes. Under the condition of same content of calcium oxalate crystals, there were no difference in irritation effect between the suspensions of raw medicinal materials and pure calcium oxalate crystals. The degree of irritation on rabbits' eyes showed undoubted quantity and irritating effect relationship with the concentrations of Calcium oxalate crystel. CONCLUSION: Calcium oxalate crystal is the irritant component in some herbal drugs which come from Araceae plants.

Comparative analysis of influence of promoters and inhibitors on in vitro available iron using two methods.

The investigation was undertaken with the objective of comparing two in vitro techniques, measuring dialyzable iron (method A) and measuring ionizable iron (method B), for iron bioavailability in a model system. The effect of the time of introduction of the additives on the available iron was also determined. FeSO4 solution was used as the reference source of iron, to which a series of enhancers (ascorbic acid, citric acid, maleic acid and tartaric acid) and inhibitors (tannic acid, calcium oxalate, oxalic acid, calcium carbonate and sodium phytate) were added individually at various concentrations, and available iron was estimated. From FeSO4 solution, 0.1% (method A) and 3.9% (method B) of iron was available. The addition of ascorbic acid, citric acid and tartaric acid increased this by 33-50%, 28-57% and 23-90%, respectively, for method A and by 15-89%, 24-78% and 24-93% for method B. Tannic acid, sodium phytate and calcium oxalate exhibited an inhibitory effect irrespective of the concentrations, while oxalic acid and calcium carbonate exhibited a dose-dependent inhibitory pattern. The iron availability analyzed by both methods showed a positive correlation with seven out of nine additives. An inverse relation was seen between the inhibitory effect of calcium carbonate and calcium oxalate and their time of introduction into the system. The overall observations showed that although absolute values varied widely, a positive correlation existed between the methods.

Effects of Tamm-Horsfall protein on the protection of MCDK cells from oxalate induced free radical injury.

Renal cell injury and fixed particle formation is one of the theories of urinary stone formation. The exposure of renal epithelial cells to oxalate ions and calcium oxalate monohydrate crystals can cause free radical generation and increase lipid peroxidation. Tamm-Horsfall protein (THP) has a protective effect on the production of free radicals in vitro. We aimed to show that THP (and its deglycosylated products, D-THP) could protect culture cells from free radical injury in vivo as well as the possible mechanism by which this is done. Exposure of Madin-Darby canine kidney (MDCK) cells to Ox resulted in a significant increase in the release LDH, NBT and MDA, as well as an increase in caspase 3 activity, all of which were further elevated when COM crystals were added. With the addition of THP at 500 nM, there was a significant decrease in the release of LDH and the production of MDA and NBT. A decrease in capase 3 activity was observed when 500 nM THP was added to the culture medium that reached 32.7% and 40.4% of inhibition in CaOx+THP and CaOx+COM+THP, respectively. THP decreased the adhesion of COM crystals to the MDCK cells but lost its effect when THP was deglycosylated. The results indicate that both Ox and COM crystals cause the release of LDH, MDA, NBT and increase the activity of capase 3 in MDCK cells. As a free radical scavenger, THP reduces the amount of free radicals and provides significant protection at a critical concentration of 500 nM. The deglycosylated THP decreased the effect of the protection of the MDCK cells from oxalate-induced injury and an increase of adhesion of the COM crystals to the MDCK cells. Therefore, the effects of THP on the protection of oxalate induced radical injury may be partly due to its intact glycosylation and its adhesion to the cell membrane.

Induction of alpha-catenin, integrin alpha3, integrin beta6, and PDGF-B by 2,8-dihydroxyadenine crystals in cultured kidney epithelial cells.

BACKGROUND: Homozygous adenine phosphoribosyltransferase (APRT) deficiency is associated with 2,8-dihydroxyadenine (DHA) nephrolithiasis. Using whole kidney RNA from Aprt knockout mice, we previously showed that the renal deposition of DHA leads to changes in the expression of genes involved in tissue injury. To determine the cellular basis for these changes, we investigated gene expression in cultured human kidney (NHK-C) and African green monkey (BSC-1) epithelial cells exposed to DHA or calcium oxalate monohydrate (COM) crystals. METHODS: First-strand cDNAs, synthesized from mRNA isolated from treated and untreated cells, were hybridized to membrane-bound cDNA arrays containing 588 genes associated with various physiological and pathological processes. Changes in gene expression were confirmed by reverse transcription PCR. RESULTS: Twenty-seven percent of the array cDNAs were expressed in untreated NHK-C cells at varying levels relative to a housekeeping gene. The expression of three adhesion molecules (alpha-catenin, integrin alpha3, and integrin beta6) and platelet-derived growth factor B (PDGF-B) was elevated following exposure of NHK-C cells to DHA. Increased expression of the adhesion molecules was also observed in BSC-1 cells, but PDGF-B expression could not be detected. COM crystals also stimulated the expression of these four genes in NHK-C cells, but the expression profile was quantitatively different compared with DHA. CONCLUSIONS: These findings suggest that DHA crystals stimulate the expression of specific genes in kidney epithelial cells and that the pathways for DHA-induced cell injury may be similar to those for COM crystals. The induction of adhesion molecules and PDGF-B may affect cell-cell or cell-matrix interactions and/or alter the actin cytoskeleton. These alterations may ultimately contribute to crystal-induced renal injury.

Phyllanthus niruri inhibits calcium oxalate endocytosis by renal tubular cells: its role in urolithiasis.

We investigated the in vitro effect of an aqueous extract of Phyllanthus niruri L. on a model of CaOx crystal endocytosis by Madin-Darby canine kidney cells. The extract exhibited a potent and effective non-concentration-dependent inhibitory effect on the CaOx crystal internalization. This response was present even at very high (pathologic) CaOx concentrations and no P. niruri L.-induced toxic effect could be detected. Biochemical analysis of culture media containing P. niruri L. did not provide any clues for the elucidation of the cellular pathways affected by this natural product. Although further studies are necessary for a better understanding of the role of P. niruri L. in urolithiasis, our findings show that this natural product could be an attractive alternative for the treatment of urinary stones.