LncRNA HOXA11-AS regulates calcium oxalate crystal-induced renal inflammation via miR-124-3p/MCP-1.

Long noncoding RNA (lncRNA) has been suggested to play an important role in a variety of diseases over the past decade. In a previous study, we identified a novel lncRNA, termed HOXA11-AS, which was significantly up-regulated in calcium oxalate (CaOx) nephrolithiasis. However, the biological function of HOXA11-AS in CaOx nephrolithiasis remains poorly defined. Here, we demonstrated that HOXA11-AS was significantly up-regulated in CaOx nephrolithiasis both in vivo and in vitro. Gain-/loss-of-function studies revealed that HOXA11-AS inhibited proliferation, promoted apoptosis and aggravated cellular damage in HK-2 cells exposed to calcium oxalate monohydrate (COM). Further investigations showed that HOXA11-AS regulated monocyte chemotactic protein 1 (MCP-1) expression in HK-2 cell model of CaOx nephrolithiasis. In addition, online bioinformatics analysis and dual-luciferase reporter assay results showed that miR-124-3p directly bound to HOXA11-AS and the 3'UTR of MCP-1. Furthermore, rescue experiment results revealed that HOXA11-AS functioned as a competing endogenous RNA to regulate MCP-1 expression through sponging miR-124-3p and that overexpression of miR-124-3p restored the inhibitory effect of proliferation, promotion effects of apoptosis and cell damage induced by HOXA11-AS overexpression. Taken together, HOXA11-AS mediated CaOx crystal-induced renal inflammation via the miR-124-3p/MCP-1 axis, and this outcome may provide a good potential therapeutic target for nephrolithiasis.

miR-155 facilitates calcium oxalate crystal-induced HK-2 cell injury via targeting PI3K associated autophagy.

Nephrolithiasis is one of the most common and highly recurrent diseases worldwide. Accumulating evidence revealed the elevated miR-155 levels both in serum and urine of nephrolithiasis patients. The aim of our research was to explore the role of miR-155 in CaOx-induced apoptosis in HK-2 cells. The expression levels of miR-155 in serum and renal tissues were quantified in 20 patients with nephrolithiasis using qRT-PCR assay. ELISA was performed to determine urinary levels of interleukin (IL)-1ß, IL-6 and tumor necrosis factor-alpha (TNF-α). Renal tubular cell model of CaOx nephrolithiasis was established to investigate the role and molelular mechanism of miR-155. Cell viability and apoptosis were assessed by MTT and flow cytometry, respectively. Immunofluoresent staining of LC3 autophagosome and western blotting were performed to evaluate the autophagic activity. Luciferase reporter assay was employed to verify the interaction between miR-155 and PI3KCA/Rheb. PI3K/Akt/mTOR signaling was further examined by western blotting. Serum and renal levels of miR-155 and inflammatory factors were significantly elevated in nephrolithiasis patients than in controls. CaOx treatment caused up-regulation of miR-155 and induced autophagy in renal tubular epithelial cells, while silencing miR-155 or inhibition of autophagy by 3-metheladenine (3-MA) ameliorated CaOx crystal-induced cell injury. PI3KCA and Rheb was identified as downstream targets of miR-155. Moreover, miR-155 activates autophagy and promotes cell injury through repressing PI3K/Akt/mTOR signaling pathway. Taken together, these findings demonstrated that miR-155 facilitates CaOx crystal-induced renal tubular epithelial cell injury via PI3K/Akt/mTOR-mediated autophagy, providing therapeutic targets for ameliorating cellular damage by CaOx crystals.

Fu-Fang-Jin-Qian-Cao herbal granules protect against the calcium oxalate-induced renal EMT by inhibiting the TGF-ß/smad pathway.

CONTEXT: Nephrolithiasis is a major public health problem worldwide and Fu-Fang-Jin-Qian-Cao granules (FFJQC) is a traditional Chinese herbal formula that is used to treat nephrolithiasis. The main component of nephrolithiasis is calcium oxalate (CaOx) and the epithelial-mesenchymal transition (EMT) shown to play a crucial role in CaOx-induced kidney injury. However, the mechanism underlying the therapeutic effect of FFJQC on the CaOx-induced renal EMT is unknown. OBJECTIVE: This study explores the therapeutic benefits and mechanism of FFJQC in oxalate-induced kidney injury. MATERIALS AND METHODS: 60 male C57BL/6 mice were used in this experiment and divided into 6 groups. A mouse kidney stone model was created by intraperitoneal injection of glyoxylate at a dose of 100 mg/kg for 6 days. The standardized FFJQC was used to treat mouse crystal kidney injury by gavage at 1.35 and 2.7 g/kg, respectively. Western blotting and immunostaining for E-cadherin, cytokeratin 18 (CK18), vimentin, smooth muscle α-actin (α-SMA) and transforming growth factor ß (TGF-ß)/Smad pathway were conducted on renal tissues. RESULTS: Following CaOx-induced kidney injury, the levels of E-cadherin and CK18 in kidney decreased, while vimentin and α-SMA levels increased. The FFJQC treatment increased the levels of E-cadherin and CK18 and decreased vimentin and α-SMA levels in varying degrees. What's more, the FFJQC reduced the expression of CaOx-induced fibrosis marker collagen II. CONCLUSION: FFJQC alleviated the CaOx-induced renal EMT and fibrosis by regulating TGF-ß/smad pathway. Therefore, the FFJQC is an important traditional Chinese medicine for the treatment of CaOx-induced renal injury and fibrosis.

Exosomes from miR-20b-3p-overexpressing stromal cells ameliorate calcium oxalate deposition in rat kidney.

Hyperoxaluria-induced calcium oxalate (CaOx) deposition is the key factor in kidney stone formation, for which adipose-derived stromal cells (ADSCs) have been used as a therapeutic treatment. Studies revealed that miR-20b-3p is down-regulated in hypercalciuric stone-forming rat kidney. To investigate whether ADSC-derived miR-20b-3p-enriched exosomes protect against kidney stones, an ethylene glycol (EG)-induced hyperoxaluria rat model and an in vitro model of oxalate-induced NRK-52E cells were established to explore the protective mechanism of miR-20b-3p. The results showed that miR-20b-3p levels were decreased following hyperoxaluria in the urine of patients and in kidney tissues from animal models. Furthermore, treatment with miR-20b-3p-enriched exosomes from ADSCs protected EG-induced hyperoxaluria rats, and cell experiments confirmed that co-culture with miR-20b-3p-enriched exosomes alleviated oxalate-induced cell autophagy and the inflammatory response by inhibiting ATG7 and TLR4. In conclusion, ADSC-derived miR-20b-3p-enriched exosomes protected against kidney stones by suppressing autophagy and inflammatory responses.

Association of sirtuin 1 gene polymorphisms with nephrolithiasis in Eastern chinese population.

Sirtuin 1 (SIRT1), an NAD+-dependent deacylase, has been identified to be associated with renal tubular inflammatory conditions and metabolic disorders, which are risk factors of nephrolithiasis. To further confirm the role of the SIRT1 in kidney stone formation, the expression of SIRT1 was analyzed based on a mouse model and the genetic polymorphisms of SIRT1 gene was compared between patients with kidney stones and controls. The calcium oxalate (CaOx) crystal-induced renal injury model was established to analyzed the expression of SIRT1 in the kidney tissue of both wild-type and ApoE(-/-) mice. And a total of 430 Eastern Chinese subjects (215 patients with nephrolithiasis and 215 age- and gender-matched controls) were recruited for the present study to investigate the associations between 6 common single nucleotide polymorphisms (SNPs) (i.e., rs10509291, rs3740051, rs932658, rs33957861, rs3818292 and rs1467568) in the SIRT1 gene and the incidence of kidney stones. Pairwise linkage disequilibrium and the haplotypes of the 6 SNPs were also analyzed. The genotypes of SIRT1 gene polymorphisms were analyzed by a Snapshot assay. Reduced expression of SIRT1 was observed in the kidney of the mice in the crystal group, revealing the potential role of SIRT1 in the nephrolithiasis. However, we did not find a significant association between the 6 SNPs of the SIRT1 gene and kidney stone formation in the Eastern Chinese population.

Efficacy of Obcordata A from Aspidopterys obcordata on Kidney Stones by Inhibiting NOX4 Expression.

Obcordata A (OA) is a polyoxypregnane glycoside derived from the Dai medicine Aspidopterys obcordata vines. This study aims to investigate the efficacy of OA on renal tubular epithelial cells exposed to calcium oxalate crystals. We incubated renal tubular cells with 28 µg·cm2 calcium oxalate crystals for 24 h with and without OA, GKT137831, phorbol-12-myristate-13-acetate (PMA), and tocopherol. The MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, microscopic examination, flow cytometry, and immunofluorescence staining revealed that calcium oxalate crystals decreased cell viability and elevated reactive oxygen species (ROS) levels. OA, GKT137831, and tocopherol protected cells and decreased ROS levels. However, OA did not exhibit direct DPPH scavenging ability. In addition, immunoblotting illustrated that OA inhibited the NOX4 (nicotinamide adenine dinucleotide phosphate oxidases 4) expression and downregulated the protein expression in the NOX4/ROS/p38 MAPK (p38 mitogen-activated protein kinase) pathway. The findings suggest that the cytoprotective and antioxidant effects of OA can be blocked by the NOX4 agonist PMA. In conclusion, OA could be used as a NOX4 inhibitor to prevent kidney stones.

Microvillar injury in renal tubular epithelial cells induced by calcium oxalate crystal and the protective role of epigallocatechin-3-gallate.

Pathogenic mechanisms of kidney stone disease remained unclear. This study investigated its initial cellular/molecular mechanisms when calcium oxalate monohydrate (COM) crystal adhered to renal tubular cells. Transmission electron microscopy revealed decreased length and density of microvilli, whereas Western blot analysis showed that whole-cell ezrin (a microvillus-stabilizing protein), not ß-actin, was decreased in COM-treated cells. Immunofluorescence staining, followed by laser-scanning confocal microscopy and subcellular fractionations, revealed decreases in both ezrin and F-/ß-actin at apical membrane. Cytoskeletal extraction by Triton X-100 showed reduced cytoskeleton-associated ezrin, consistent with colocalization data of ezrin/F-actin. Thr567-phosphorylated ezrin and RhoA increased in COM-treated cells. A protein oxidation blot assay showed an increase in oxidized proteins in COM-treated cells that could be prevented by epigallocatechin-3-gallate (EGCG), which also preserved the whole-cell ezrin level, stabilized apical membrane ezrin/F-actin colocalization, and maintained microvillar structure in COM-treated and H2O2-treated cells. Our data clearly demonstrated the reduction of ezrin and actin expression at the apical membrane of COM-treated cells, most likely because of oxidative stress, which could be prevented by EGCG. These findings provide a novel approach to better understanding of the pathogenesis of kidney stone disease in its initial phase and offer potential preventive strategy against microvillar injury induced by COM crystals in patients with kidney stones.-Fong-ngern, K., Vinaiphat, A., Thongboonkerd, V. Microvillar injury in renal tubular epithelial cells induced by calcium oxalate crystal and the protective role of epigallocatechin-3-gallate.

Efficacy of Hydroxy-L-proline (HYP) analogs in the treatment of primary hyperoxaluria in Drosophila Melanogaster.

BACKGROUND: Substrate reduction therapy with analogs reduces the accumulation of substrates by inhibiting the metabolic pathways involved in their biosynthesis, providing new treatment options for patients with primary hyperoxalurias (PHs) that often progress to end-stage renal disease (ESRD). This research aims to evaluate the inhibition efficacy of Hydroxy-L-proline (HYP) analogs against calcium oxalate (CaOx) crystal formation in the Drosophila Melanogaster (D. Melanogaster) by comparing them with Pyridoxine (Vitamin B6). METHODS: Three stocks of Drosophila Melanogaster (W118, CG3926 RNAi, and Act5C-GAL4/CyO) were utilized. Two stocks (CG3926 RNAi and Act5C-GAL4 /CyO) were crossed to generate the Act5C > dAGXT RNAi recombinant line (F1 generation) of D. Melanogaster which was used to compare the efficacy of Hydroxy-L-proline (HYP) analogs inhibiting CaOx crystal formation with Vitamin B6 as the traditional therapy for primary hyperoxaluria. RESULTS: Nephrolithiasis model was successfully constructed by downregulating the function of the dAGXT gene in D. Melanogaster (P-Value = 0.0045). Furthermore, the efficacy of Hydroxy-L-proline (HYP) analogs against CaOx crystal formation was demonstrated in vivo using D. Melanogaster model; the results showed that these L-Proline analogs were better in inhibiting stone formation at very low concentrations than Vitamin B6 (IC50 = 0.6 and 1.8% for standard and dietary salt growth medium respectively) compared to N-acetyl-L-Hydroxyproline (IC50 = 0.1% for both standard and dietary salt growth medium) and Baclofen (IC50 = 0.06 and 0.1% for standard and dietary salt growth medium respectively). Analysis of variance (ANOVA) also showed that Hydroxy-L-proline (HYP) analogs were better alternatives for CaOx inhibition at very low concentration especially when both genetics and environmental factors are intertwined (p < 0.0008) for the dietary salt growth medium and (P < 0.063) for standard growth medium. CONCLUSION: Addition of Hydroxy-L-Proline analogs to growth medium resulted in the reduction of CaOx crystals formation. These analogs show promise as potential inhibitors for oxalate reduction in Primary Hyperoxaluria.

The mechanism of the preventive effect of Shen'an capsule on the calcium oxalate crystal-induced early renal injury based on metabolomics.

Kidney stone disease is a worldwide metabolism-associated disorder with a high incidence of renal dysfunction. However, effective methods to prevent crystalline nephropathy are still lacking owing to the absence of aetiological research. Shen'an (SA) capsules are prepared from Chinese medicinal compounds and are considered a promising treatment for the prevention of crystal-induced renal injury. In this study, 24 mice were randomly divided into four groups: saline, oxalate, SA-treated (via preventive administration) and SA-only groups. A metabolomics analysis based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was performed to explore the plasma metabolic profiles among the different groups. The amount of crystal deposition and the decline in kidney function were significantly alleviated by the use of SA capsule. A total of 24 metabolites that showed a reversal trend following SA capsule administration were identified as plasma biomarkerss of the preventive effects of SA capsules on crystal-induced renal injury. Most of these metabolites were involved in the metabolisms of lipid metabolism, energy metabolism, glutathione metabolism and vitamin metabolism. In conclusion, SA capsules exert a preventive effect in mice with crystal-induced kidney injury via the regulation of multiple metabolic pathways.

Aluminum citrate blocks toxicity of calcium oxalate crystals by preventing binding with cell membrane phospholipids.

BACKGROUND/AIMS: Renal damage from ethylene glycol and primary hyperoxaluria is linked to accumulation of calcium oxalate monohydrate (COM) crystals in the renal proximal tubule (PT). In vitro studies have shown that aluminum citrate (AC), uniquely among citrate salts, blocks COM cytotoxicity to tubular cells. These studies were designed to evaluate the interaction of COM with membrane phospholipids and the ability of AC to reduce COM toxicity by interfering with this interaction. METHODS: Interaction of COM with phospholipids was assessed using differential scanning calorimetric analysis of structural changes in specific liposomes. Interaction of COM with cell membranes was studied by measuring binding of radiolabeled crystals by human PT (HPT) cells. RESULTS: Analysis of liposomes prepared from phosphatidylserine (PS) or phosphatidylcholine (PC) showed that COM interfered with the gel-liquid transition of PS liposomes, but not that of PC liposomes. AC reversed the COM-induced changes in liposomal structure. AC inhibited the binding of [(14)C]-COM by HPT cells in a concentration-dependent manner. AC blocked COM binding by interacting with the crystal surface and not the cell membrane. CONCLUSION: These results indicate that AC blocks the binding of COM by PT cells, and consequently its cytotoxicity, by attaching to the surface of the crystal. Thus, AC, or a related compound that works by the same mechanism, could be a useful adjunct therapy to reduce the renal damage produced by severe hyperoxaluria.

Oral Reference Dose for ethylene glycol based on oxalate crystal-induced renal tubule degeneration as the critical effect.

Several risk assessments have been conducted for ethylene glycol (EG). These assessments identified the kidney as the primary target organ for chronic effects. None of these assessments have incorporated the robust database of species-specific toxicokinetic and toxicodynamic studies with EG and its metabolites in defining uncertainty factors used in reference value derivation. Pertinent in vitro and in vivo studies related to one of these metabolites, calcium oxalate, and its role in crystal-induced nephropathy are summarized, and the weight of evidence to establish the mode of action for renal toxicity is reviewed. Previous risk assessments were based on chronic rat studies using a strain of rat that was later determined to be less sensitive to the toxic effects of EG. A recently published 12-month rat study using the more sensitive strain (Wistar) was selected to determine the point of departure for a new risk assessment. This approach incorporated toxicokinetic and toxicodynamic data and used Benchmark Dose methods to calculate a Human Equivalent Dose. Uncertainty factors were chosen, depending on the quality of the studies available, the extent of the database, and scientific judgment. The Reference Dose for long-term repeat oral exposure to EG was determined to be 15 mg/kg bw/d.

Genetic Background but Not Intestinal Microbiota After Co-Housing Determines Hyperoxaluria-Related Nephrocalcinosis in Common Inbred Mouse Strains.

Calcium oxalate (CaOx) crystal formation, aggregation and growth is a common cause of kidney stone disease and nephrocalcinosis-related chronic kidney disease (CKD). Genetically modified mouse strains are frequently used as an experimental tool in this context but observed phenotypes may also relate to the genetic background or intestinal microbiota. We hypothesized that the genetic background or intestinal microbiota of mice determine CaOx crystal deposition and thus the outcome of nephrocalcinosis. Indeed, Casp1-/-, Cybb-/- or Casp1-/-/Cybb-/- knockout mice on a 129/C57BL/6J (B6J) background that were fed an oxalate-rich diet for 14 days did neither encounter intrarenal CaOx crystal deposits nor nephrocalcinosis-related CKD. To test our assumption, we fed C57BL/6N (B6N), 129, B6J and Balb/c mice an oxalate-rich diet for 14 days. Only B6N mice displayed CaOx crystal deposits and developed CKD associated with tubular injury, inflammation and interstitial fibrosis. Intrarenal mRNA expression profiling of 64 known nephrocalcinosis-related genes revealed that healthy B6N mice had lower mRNA levels of uromodulin (Umod) compared to the other three strains. Feeding an oxalate-rich diet caused an increase in uromodulin protein expression and CaOx crystal deposition in the kidney as well as in urinary uromodulin excretion in B6N mice but not 129, B6J and Balb/c mice. However, backcrossing 129 mice on a B6N background resulted in a gradual increase in CaOx crystal deposits from F2 to F7, of which all B6N/129 mice from the 7th generation developed CaOx-related nephropathy similar to B6N mice. Co-housing experiments tested for a putative role of the intestinal microbiota but B6N co-housed with 129 mice or B6N/129 (3rd and 6th generation) mice did not affect nephrocalcinosis. In summary, genetic background but not the intestinal microbiome account for strain-specific crystal formation and, the levels of uromodulin secretion may contribute to this phenomenon. Our results imply that only littermate controls of the identical genetic background strain are appropriate when performing knockout mouse studies in this context, while co-housing is optional.

Ursolic acid treats renal tubular epithelial cell damage induced by calcium oxalate monohydrate via inhibiting oxidative stress and inflammation.

Ursolic acid (UA) has been proved to have antioxidant and anti-inflammatory effects. However, it is not clear whether it has a protective impact on kidney damage induced by crystals of calcium oxalate monohydrate (COM). This work aimed to make clear the potential mechanism of UA protecting COM-induced kidney damage. The results manifested that high- and low-dose UA reduced COM crystals in COM rats' kidney, down-regulated urea, creatinine, and neutrophil gelatinase-associated lipocalin (NGAL) levels in rat plasma, declined kidney tissue and HK-2 cell apoptosis, inhibited Bax expression but elevated Bcl-2 expression. Additionally, UA alleviated renal fibrosis in COM rats, repressed α-SMA and collagen I protein expressions in the kidney and COM rats' HK-2 cells, depressed COM-induced oxidative damage in vivo and in vitro via up-regulating Nrf2/HO-1 pathway, up-regulated SOD levels and reduced MDA levels, down-regulated TNF-α, IL-1ß, and IL-6 levels in vivo and in vitro via suppressing activation of TLR4/NF-κB pathway. In summary, the results of this study suggest that COM-induced renal injury can be effectively improved via UA, providing powerful data support for the development of effective clinical drugs for renal injury in the future.

6,7-Dihydroxycoumarin ameliorates crystal-induced necroptosis during crystal nephropathies by inhibiting MLKL phosphorylation.

AIMS: Mineralization of crystalline particles and the formation of renal calculi contribute to the pathogenesis of crystal nephropathies. Several recent studies on the biology of crystal handling implicated intrarenal crystal deposition-induced necroinflammation in their pathogenesis. We hypothesized that 6,7-dihydroxycoumarin (DHC) inhibit intrarenal crystal cytotoxicity and necroinflammation, and ameliorate crystal-induced chronic kidney disease (CKD). MAIN METHODS: An unbiased high content screening coupled with fluorescence microscopy was used to identify compounds that inhibit CaOx crystal cytotoxicity. The ligand-protein interactions were identified using computational models e.g. molecular docking and molecular dynamics simulations. Furthermore, mice and rat models of oxalate-induced CKD were used for in-vivo studies. Renal injury, crystal deposition, and fibrosis were assessed by histology analysis. Western blots were used to quantify the protein expression. Data were expressed as boxplots and analyzed using one way ANOVA. KEY FINDINGS: An unbiased high-content screening in-vitro identified 6,7-DHC as a promising candidate. Further, 6,7-DHC protected human and mouse cells from calcium oxalate (CaOx) crystal-induced necroptosis in-vitro as well as mice and rats from oxalate-induced CKD in either preventive or therapeutic manner. Computational modeling demonstrated that 6,7-DHC interact with MLKL, the key protein in the necroptosis machinery, and inhibit its phosphorylation by ATP, which was evident in both in-vitro and in-vivo analyses. SIGNIFICANCE: Together, our results indicate that 6,7-DHC possesses a novel pharmacological property as a MLKL inhibitor and could serve as a lead molecule for further development of coumarin-based novel MLKL inhibitors. Furthermore, our data identify 6,7-DHC as a novel therapeutic strategy to combat crystal nephropathies.

Vitexin exerts protective effects against calcium oxalate crystal-induced kidney pyroptosis in vivo and in vitro.

BACKGROUND: Nephrolithiasis is a common urinary disease with a high recurrence rate of secondary stone formation. Several mechanisms are involved in the onset and recurrence of nephrolithiasis, e.g., oxidative stress, inflammation, apoptosis, and epithelial-mesenchymal transition (EMT). Vitexin, a flavonoid monomer derived from medicinal plants that exert many biological effects including anti-inflammatory and anticancer effects, has not been investigated in nephrolithiasis studies. Moreover, pyroptosis, a form of programmed cell death resulting from inflammasome-associated caspase activation, has not been studied in mice with nephrolithiasis. PURPOSE: We aimed to investigate the protective effect and underlying mechanisms of vitexin in nephrolithiasis, and the related role of pyroptosis in vivo and in vitro. METHODS: Mouse models of nephrolithiasis were established via intraperitoneal injection of glyoxylate, and cell models of tubular epithelial cells and macrophages were established using calcium oxalate monohydrate (COM). Crystal deposition and kidney tissue injury were evaluated by hematoxylin and eosin, and von Kossa staining. Renal oxidative stress indexes including malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and catalase (CAT), were analyzed. The renal expression of interleukin-1 beta (IL-1ß), gasdermin D (GSDMD), osteopontin (OPN), CD44, and monocyte chemotactic protein 1 (MCP-1), and EMT-related proteins in renal tubular epithelial cells was assessed. Cell viability and the apoptosis ratio were evaluated. RESULTS: In vivo, vitexin alleviated crystal deposition and kidney tissue injury, and decreased the level of MDA, and increased the levels of SOD, GSH, and CAT. Vitexin also reduced the levels of the pyroptosis-related proteins GSDMD, NLRP3, cleaved caspase-1, and mature IL-1ß, which were elevated in mice with nephrolithiasis, and repressed apoptosis and the expression of OPN and CD44. Moreover, vitexin mitigated F4/80-positive macrophage infiltration and MCP-1 expression in the kidneys. Furthermore, an in vitro study showed that vitexin increased the viability of HK-2 cells and THP-1-derived macrophages, which was impaired by treatment with COM crystals, decreased the medium lactate dehydrogenase (LDH) level, and inhibited the expression of pyroptosis-related proteins in HK-2 cells and macrophages. Vitexin repressed EMT of HK-2 cells, with increased expression of pan-cytokeratin (Pan-ck) and decreased expression of Vimentin and alpha-smooth muscle actin (α-SMA), and downregulated the Wnt/ß-catenin pathway. Moreover, vitexin suppressed tumor necrosis factor-α (TNF-α) and IL-1ß mRNA expression, which was upregulated by COM in macrophages. CONCLUSION: Vitexin exerts protective effects against nephrolithiasis by inhibiting pyroptosis activation, apoptosis, EMT, and macrophage infiltration. In addition, GSDMD-related pyroptosis mediates nephrolithiasis.

Downregulation of inflammatory mediators by ethanolic extract of Bergenia ligulata (Wall.) in oxalate injured renal epithelial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: In the Indian traditional system of medicine, Bergenia ligulata (Wall.) Engl. has been used for treatment of urolithiasis. Its efficacious nature has led to its incorporation in various commercial herbal formulations such as Cystone and Neeri which are prescribed for kidney related ailments. AIM OF THE STUDY: To assess whether ethanolic extract of B. ligulata can mitigate the cascade of inflammatory responses that cause oxidative stress and ultimately cell death in renal epithelial cells exposed to hyperoxaluric conditions. MATERIAL AND METHODS: Bioactivity guided fractionation using solvents of varying polarities was employed to evaluate the potential of the extracts of B. ligulata to inhibit the crystallization process. Modulation of crystal morphology was visualized through Scanning electron microscopy (SEM) analysis. Cell death was assessed using flow cytometry based assays. Alteration in the inflammatory mediators was evaluated using real time PCR and immunocytochemistry. Phytochemical characterization of the ethanolic extract was carried out using FTIR, LC-MS and GC-MS. RESULTS: Bioactivity guided fractionation for the assessment of antilithiatic activity revealed dose dependent inhibition of nucleation and aggregation process of calcium oxalate crystals in the presence of various extracts, however ethanolic extract showed maximum inhibition and was chosen for further experiments. Studies on renal epithelial NRK-52E cells showed, cytoprotective efficacy of B. ligulata extract against oxalate injury. SEM anaysis further revealed the potential of the extract to modulate the crystal structure and adhesion to renal cell surface. Exposure of the renal cells to the extract led to conversion of the calcium oxalate monohydrate (COM) crystals to the less injurious calcium oxalate dihydrate (COD) form. Expression analysis for oxidative stress and inflammatory biomarkers in NRK-52E cells revealed up-regulation of Mitogen activated protein kinase (MAPK), Osteopontin (OPN) and Nuclear factor- ĸB (NF-ĸB), in response to calcium oxalate insult; which was drastically reduced in the presence of B. ligulata extract. Flow cytometric evaluation pointed to caspase 3 mediated apoptotic cell death in oxalate injured cells, which was attenuated by B. ligulata extract. CONCLUSION: Considering the complex multifactorial etiology of urolithiasis, ethanolic extract from B. ligulata can be a promising option for the management of kidney stones, as it has the potential to limit inflammation and the subsequent cell death.

Protective Effect of Degraded Porphyra yezoensis Polysaccharides on the Oxidative Damage of Renal Epithelial Cells and on the Adhesion and Endocytosis of Nanocalcium Oxalate Crystals.

The protective effects of Porphyra yezoensis polysaccharides (PYPs) with molecular weights of 576.2 (PYP1), 105.4 (PYP2), 22.47 (PYP3), and 3.89 kDa (PYP4) on the oxidative damage of human kidney proximal tubular epithelial (HK-2) cells and the differences in adherence and endocytosis of HK-2 cells to calcium oxalate monohydrate crystals before and after protection were investigated. Results showed that PYPs can effectively reduce the oxidative damage of oxalic acid to HK-2 cells. Under the preprotection of PYPs, cell viability increased, cell morphology improved, reactive oxygen species levels decreased, mitochondrial membrane potential increased, S phase cell arrest was inhibited, the cell apoptosis rate decreased, phosphatidylserine exposure reduced, the number of crystals adhered to the cell surface reduced, but the ability of cells to endocytose crystals enhanced. The lower the molecular weight, the better the protective effect of PYP. The results in this article indicated that PYPs can reduce the risk of kidney stone formation by protecting renal epithelial cells from oxidative damage and reducing calcium oxalate crystal adhesion, and PYP4 with the lowest molecular weight may be a potential drug for preventing kidney stone formation.

Proteome changes in human monocytes upon interaction with calcium oxalate monohydrate crystals.

Monocytic infiltration in renal interstitium is commonly found surrounding the site of calcium oxalate (CaOx) crystal deposition in the kidney. Monocytes are supposed to eliminate the deposited crystals. However, effects of CaOx crystals on the infiltrating monocytes remain unknown. Therefore, this study investigated the altered cellular proteome of human monocytes in response to interaction with CaOx monohydrate (COM) crystals. After 24-h culture with or without 100 microg/mL COM crystals, U937 cells were harvested and subjected to 2-DE analysis with Deep Purple fluorescence staining (n = 5 gels/group; each was derived from independent culture). Spot matching, quantitative intensity analysis, and statistics revealed 22 differentially expressed proteins (9 up-regulated and 13 down-regulated proteins), which were successfully identified by Q-TOF MS and MS/MS analyses, including those involved in cell cycle, cellular structure, carbohydrate metabolism, lipid metabolism, mRNA processing, and protein synthesis, stabilization, and degradation. Randomly selected changes [up-regulated ALG-2 interacting protein 1 (Alix), elongation factor-2 (EF-2), and down-regulated beta-actin] were confirmed by Western blot analysis. Our data may help to understand how monocytes interact with COM crystals. These processes are proposed to cause subsequent inflammatory response in kidney stone disease through oxidative stress pathway(s).

[Comparisons of crystal form of raphides to toxicity raphides in four poisonous herbs of Araceae family].

OBJECTIVE: To compare the crystal form with the toxicity intensitity of raphides in four poisonous herbs of Araceae family. METHOD: The four kinds of raphides were extracted and isolated from Pinellia ternate, P. pedatisecta, Arisaema amurense and Typhonium giganteum. These raphides were observed with scanning electron microscopy (SEM), and the elements were analyzed with X-ray photoelectron spectroscopy. Infrared spectrum was used for detecting the functional groups. Toxic intensities of the four kinds of raphides were detected by mice acute toxicity experiment, and the value of LD0 were from caculated by Bliss rule. RESULT: The raphides in the four plants have the similar crystal form. Observation with SEM showed a pointed and blunt end, and a long groove and barbs on a raphide. The raphides in P. ternate and P. pedatisecta were sharper than that in other two, respectively. The results of X-ray diffraction, photoelectric spectra showed that the major component of raphides was calcium oxalate monohydrate, and also showed the elements of N and S existing. Infrared spectra showed the raphides contained functional groups of -COOH and -NH2. These results illustrated that the calcium oxalate monohydrate was not the only component of the raphide. The raphides could produce severe toxic reactions. LD50 values of P. ternate, P. pedatisecta, A. amurense and T. giganteum were 14.78, 14.11, 16.02 and 18.90 mg x kg(-1) (ip), respectively. The corresponding LD50 values of crude drugs were all above 3000 mg x kg(-1) (ip). The toxicity of raphides was 200 times of crude drugs'. CONCLUSION: The raphides in P. ternate and P. pedatisecta, A. amurense and T. giganteum were their common poisonous factor.

Potentially Pathogenic Calcium Oxalate Dihydrate and Titanium Dioxide Crystals in the Alzheimer's Disease Entorhinal Cortex.

Knowing that Alzheimer's disease (AD) nucleates in the entorhinal cortex (EC), samples of 12 EC specimens were probed for crystals by a protocol detecting fewer than 1/5000th of those present. Of the 61 crystals found, 31 were expected and 30 were novel. Twenty-one crystals of iron oxides and 10 atherosclerosis-associated calcium pyrophosphate dihydrate crystals were expected and found. The 30 unexpected crystals were NLRP3-inflammasome activating calcium oxalate dihydrate (12) and titanium dioxide (18). Their unusual distribution raises the possibility that some were of AD origination sites.