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.

AhR activation attenuates calcium oxalate nephrocalcinosis by diminishing M1 macrophage polarization and promoting M2 macrophage polarization.

Calcium oxalate (CaOx) crystal can trigger kidney injury, which contributes to the pathogenesis of nephrocalcinosis. The phenotypes of infiltrating macrophage may impact CaOx-mediated kidney inflammatory injury as well as crystal deposition. How aryl hydrocarbon receptor (AhR) regulates inflammation and macrophage polarization is well understood; however, how it modulates CaOx nephrocalcinosis remains unclear. Methods: Mice were intraperitoneally injected with glyoxylate to establish CaOx nephrocalcinosis model with or without the treatment of AhR activator 6-formylindolo(3,2-b)carbazole (FICZ). Positron emission tomography computed tomography (PET-CT) imaging, Periodic acid-Schiff (PAS) staining, and polarized light optical microscopy were used to evaluate kidney injury and crystal deposition in mice kidney. Western blotting, immunofluorescence, chromatin immunoprecipitation, microRNA-fluorescence in situ hybridization, and luciferase reporter assays were applied to analyze polarization state and regulation mechanism of macrophage. Results: AhR expression was significantly upregulated and negatively correlated with interferon-regulatory factor 1 (IRF1) and hypoxia inducible factor 1-alpha (HIF-1α) levels in a murine CaOx nephrocalcinosis model following administration of FICZ. Moreover, AhR activation suppressed IRF1 and HIF-1α levels and decreased M1 macrophage polarization in vitro. In terms of the mechanism, bioinformatics analysis and chromatin immunoprecipitation assay confirmed that AhR could bind to miR-142a promoter to transcriptionally activate miR-142a. In addition, luciferase reporter assays validated that miR-142a inhibited IRF1 and HIF-1α expression by directly targeting their 3'-untranslated regions. Conclusions: Our results indicated that AhR activation could diminish M1 macrophage polarization and promote M2 macrophage polarization to suppress CaOx nephrocalcinosis via the AhR-miR-142a-IRF1/HIF-1α pathway.

Curcumin ameliorates glyoxylate-induced calcium oxalate deposition and renal injuries in mice.

BACKGROUND: Nephrolithiasis is one of the most common and frequent urologic diseases worldwide. Several pathophysiological mechanisms are involved in stone formation, including oxidative stress, inflammation, apoptosis, fibrosis and autophagy. Curcumin, the predominant active component of turmeric, has been shown to have pleiotropic biological and pharmacological properties, such as antioxidant, anti-inflammatory and antifibrotic effects. PURPOSE: The current study proposed to systematically investigate the protective effects and the underlying mechanisms of curcumin in a calcium oxalate (CaOx) nephrolithiasis mouse model. METHODS: The animal model was established in male C57BL/6 mice by successive intraperitoneal injection of glyoxylate (100 mg/kg) for 1 week. Curcumin was orally given to mice 7 days before the injection of glyoxylate and for a total of 14 days at 50 mg/kg or 100 mg/kg. Bilateral renal tissue was harvested and processed for oxidative stress index detection, histopathological examinations and other analyses. RESULTS: Coadministration of curcumin could significantly reduce glyoxylate-induced CaOx deposition and simultaneous tissue injury in mouse kidneys. Meanwhile, curcumin alleviated the oxidative stress response via reducing MDA content and increasing SOD, CAT, GPx, GR and GSH levels in this animal model. Moreover, treatment with curcumin significantly inhibited apoptosis and autophagy induced by hyperoxaluria. Curcumin also attenuated the high expression of IL-6, MCP-1, OPN, CD44, α-SMA, Collagen I and collagen fibril deposition, which were elevated by hyperoxaluria. Furthermore, the results revealed that both the total expression and nuclear accumulation of Nrf2, as well as its main downstream products such as HO-1, NQO1 and UGT, were decreased in the kidneys of mice in the crystal group, while treatment with curcumin could rescue this deterioration. CONCLUSION: Curcumin could significantly alleviate CaOx crystal deposition in the mouse kidney and the concurrent renal tissue injury. The underlying mechanism involved the combination of antioxidant, anti-apoptotic, inhibiting autophagy, anti-inflammatory, and antifibrotic activity and the ability to decrease expression of OPN and CD44 through the Nrf2 signaling pathway. The pleiotropic antilithic properties, combined with the minimal side effects, make curcumin a good potential choice to prevent and treat new or recurrent nephrolithiasis.

Sirtuin 3 suppresses the formation of renal calcium oxalate crystals through promoting M2 polarization of macrophages.

This study aims to verify whether the inhibitory effect of Sirtuin 3 (SIRT3) on the formation of renal calcium oxalate crystals was mediated through promoting macrophages (Mϕs) polarization. Identification and quantification of M1 and M2 monocytes were performed using fluorescence-activated cell sorting analysis. SIRT3 protein level and forkhead box O1 (FOXO1) acetylation level were measured using western blot analysis. Cell apoptosis of HK-2 was detected by flow cytometry. Mouse kidney tissues were subjected to Von Kossa staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and immunohistochemical staining for detection of kidney crystals deposition, apoptosis, and expression of crystal-related molecules, respectively. The results showed that human peripheral blood monocytes from patients with kidney stone (KS) exhibited decreased M2 monocytes percentage and SIRT3 expression, whereas increased FOXO1 acetylation compared with the normal controls. In vitro assay revealed that SIRT3 overexpression in bone marrow-derived M0/M1/M2 Mϕs induced M2 polarization and decreased FOXO1 acetylation. Furthermore, FOXO1 knockdown reversed SIRT3-mediated induction of M2 polarization and inhibition of HK-2 (human proximal tubular cell line) apoptosis. Further in vivo experiments demonstrated that SIRT3-overexpressing Mϕs transfusion not only induced M2 polarization, but also alleviated inflammation, apoptosis, and crystals deposition in glyoxylate-induced KS mice. In conclusion, SIRT3 suppresses formation of renal calcium oxalate crystals through promoting M2 polarization via deacetylating FOXO1.

Genetic differences in C57BL/6 mouse substrains affect kidney crystal deposition.

We previously established an experimental model of calcium oxalate crystal deposition in the mouse kidney using C57BL/6 mice. C57BL/6J (B6J) and C57BL/6N (B6N) are two core substrains of C57BL/6 mice. B6J and B6N substrains have approximately the same genomic sequence. However, in whole-genome analyses, substrains have slight genetic differences in some genes. In this study, we used these substrains as kidney crystal formation models and compared their genetic backgrounds to elucidate the pathogenic mechanisms of kidney stone formation. Eight-week-old male B6J and B6N mice (n = 15 in each group) were administered 80 mg/kg glyoxylate for 12 days, and the amount of kidney crystal depositions was compared. The expression levels of six genes (Snap29, Fgf14, Aplp2, Lims1, Naaladl2, and Nnt) were investigated by quantitative polymerase chain reaction, and the protein levels were evaluated by western blotting and immunohistochemistry. The amount of kidney crystal depositions was significantly higher in B6J mice than in B6N mice on days 6 and 12. The expression of nicotinamide nucleotide transhydrogenase (Nnt) gene was significantly lower in B6J mice than in B6N mice. The expression of Nnt protein was observed only in B6N mice, and preferential high expression was seen in renal tubular epithelial cells. The results of this study provide compelling evidence that differences in mouse substrains affect kidney crystal deposition and that the absence of Nnt protein could be involved in crystal formation in B6J mice.

Inhalation of hydrogen gas ameliorates glyoxylate-induced calcium oxalate deposition and renal oxidative stress in mice.

The aim of this study is to evaluate the protective effect and underlying mechanism of hydrogen gas (H2) to glyoxylate induced renal calcium oxalate (CaOx) crystal deposition in mice. In present work, rodent renal CaOx crystal deposition model was introduced by intra-abdominal injection of glyoxylate (100 mg/kg/d) for 5 days. Two days before administration of glyoxylate, inhalation of H2 for 30 min per day was initiated and continued for 7 days. By the end of the study, the samples of 24 hours urine, serum and renal tissue were collected for biochemical and pathological assay. According to levels of urine calcium excretion, renal calcium deposition, a serum excretion of kidney injury molecule-1 (KIM-1) assay and a TUNEL assay, inhalation of H2 could successfully decrease the CaOx crystallizations and protect against renal injury. Crystal deposition in the kidneys is associated with oxidative stress, which was indicated by increased levels of renal malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) and decreased activities of superoxide dismutase (SOD), glutathione (GSH) and catalase (CAT). These effects were reversed by a high-dose H2 pretreatment. The renal expressions of osteopontin (OPN), CD44, monocyte chemoattractant protein-1 (MCP-1) and interleukin-10 (IL-10) were markedly increased in glyoxylate-treated mice, and H2 significantly attenuated the increase of OPN, CD44 and MCP-1 but upregulated the expression of IL-10. Our findings demonstrate that inhalation of H2 reduces renal crystallization, renal oxidative injury and inflammation and it may be a candidate agent with few adverse effects for prevention of nephrolithiasis.

The styrene metabolite, phenylglyoxylic acid, induces striatal-motor toxicity in the rat: influence of dose escalation/reduction over time.

Exposure to the industrial solvent, styrene, induces locomotor and cognitive dysfunction in rats, and parkinsonian-like manifestations in man. The antipsychotic, haloperidol (HP), well known to induce striatal toxicity in man and animals, and styrene share a common metabolic pathway yielding p-fluoro phenylglyoxylic acid and phenylglyoxylic acid (PGA), respectively. Using an exposure period of 30 days and the vacous chewing movement (VCM) model as an expression of striatal-motor toxicity, we found that incremental PGA dosing (220-400 mg/kg) significantly increased VCMs up to day 25, but decreased to control levels shortly after reaching maximum dose. However, a diminishing dose of PGA (400-200 mg/kg) did not evoke an immediate worsening of VCMs but precipitated a significant increase in VCMs following dosage reduction to 200 mg/kg on day 22. PGA exposure, therefore, compromises striatal-motor function that is especially sensitive to changes in exposure dose. Longer alternating dose exposure studies are needed to establish whether motor dysfunction is progressive in severity or longevity. These findings are of significance for the environmental toxicology of styrene in the chemical industry.

Glyoxylate induces renal tubular cell injury and microstructural changes in experimental mouse.

Crystal formation in mice could not be induced either by the administration of ethylene glycol or by glycolate. To clarify the reasons for the difference among these oxalate precursors in mice, we studied renal tubular epithelial injury by immunohistochemical staining of oxidative stress and observing microstructures. Daily intra-abdominal injection of saline solution [10 ml/(kg day)], ethylene glycol[(48.3 mmol/(kg day)], glycolate [1.31 mmol/(kg day)], and glyoxylate [1.35 mmol/(kg day)] into C57BL/6 male mice (8 weeks) was performed for 7 days. Immunohistochemical staining of superoxide dismutase (SOD) and malondialdehyde (MDA), and transmission electron microscopy (TEM) of renal tubular epithelial cells were performed to observe oxidative stress and morphological changes, respectively. Decreased SOD and increased MDA were shown only in glyoxylate-treated mouse kidneys. The TEM study with glyoxylate-treated mouse kidneys demonstrated that the internal structure of mitochondria in renal tubular cells underwent destruction and vacuolization, and microvilli density decreased. These changes in renal tubular cells were located in the crystal-forming area. However, such changes were not detected in the other groups. Each precursor of oxalate induces different changes in renal epithelial cells regarding oxidative stress and the microstructural changes. It is suggested that calcium oxalate crystal formation requires cell injury and morphological changes of renal epithelial tubular cells induced by glyoxylate administration in the mouse kidney.

Irritancy and sensitization potential of glyoxylic acid.

Glyoxylic acid, a small dicarboxylic acid, has been detected at measurable levels in the atmosphere and is suspected to be present in indoor air environments. It is generated through the ozonolysis of several high volume production compounds that are commonly found indoors. Glyoxylic acid was tested in a combined irritancy and local lymph node assay (LLNA). It tested positive in the LLNA with an EC3 value of 5.05%. Significant increases were observed in the B220+cell population in the draining lymph nodes. No changes were identified in the IgE+B220+ cell population in the draining lymph nodes or total serum IgE levels; this suggests that glyoxylic acid functions as a T-cell-mediated contact sensitizer. Exposure to volatile organic compounds (VOC), similar to glyoxylic acid, emitted from building materials, cleaning formulations or other consumer products, and /or indoor chemistry have been linked to adverse health effects. These results may provide an explanation for some of adverse health effects associated with indoor air exposure.

Calcium oxalate, and not other metabolites, is responsible for the renal toxicity of ethylene glycol.

Ethylene glycol (EG) is nephrotoxic due to its metabolism. Many studies suggest that the toxicity is due to oxalate accumulation, but others have conversely suggested that toxicity results from effects of metabolites such as glycolaldehyde or glyoxylic acid on proximal tubule cells. In vivo studies have indicated that accumulation of calcium oxalate monohydrate (COM) corresponds closely with development of toxicity in renal tissue. The present studies were therefore designed to clarify the roles of various metabolites in the mechanism for EG toxicity in vitro by comparing the relative cytotoxicity of EG metabolites using three measures of cell death, ethidium homodimer uptake, lactate dehydrogenase (LDH) release and the conversion of the tetrazolium salt XTT to a colorimetric dye. Human proximal tubule cells in culture were incubated in physiologic buffers for 6h at 37 degrees C with COM (147-735microg/ml, an oxalate equivalence of 1-5mM), glycolate (5-25mM), glyoxylate (0.2-5mM) and glycolaldehyde (0.2-2mM). To assess the effects of acidity on the cytotoxicity, incubations were carried out at pH 6-7.4. The results show that COM dose-dependently increased LDH release and ethidium homodimer uptake, while the other metabolites did not. Conversely, COM had no effect on the XTT assay, while high concentrations of glycolaldehyde and glyoxylate decreased XTT activity, but the latter only at acidic pH. The correlation between the uptake of ethidium homodimer and the release of LDH suggest that COM is cytotoxic to human kidney cells in culture, while the XTT assay does not validly measure cytotoxicity in this system. These results indicate that COM, and not glyoxylate or glycolaldehyde, is the toxic metabolite responsible for the acute tubular necrosis and renal failure that is observed in EG-poisoned patients.

Effects of ethylene glycol and metabolites on in vitro development of rat embryos during organogenesis.

The aim of this study was to investigate in vitro the relative impact of ethylene glycol, a major industrial chemical, and its individual metabolites on the embryonic development of rats. Rat whole embryos were exposed for 48 h (day 9.5-11.5 of gestation) to ethylene glycol (EG) and its metabolites glycolaldehyde (GAl), glycolic acid (GA), glyoxylic acid (GXA), glyoxale (GXAl) and oxalic acid (OXA) at increasing concentrations. Embryotoxic concentrations were achieved within the following range: ethylene glycol (100-200 mM), glycolic acid (3 mM), glyoxal (6 mM), oxalic acid (1-3 mM), glyoxylic acid (0.3-1 mM), glycolaldehyde (0.1-0.2 mM). The pattern of dysmorphogenesis with all compounds including EG showed a general embryotoxicity with diffusely distributed cell necroses with no specific target tissues selectively affected. The results obtained in this study emphasize the hypothesis that the metabolites and not ethylene glycol itself are responsible for the embryotoxicity of ethylene glycol in rats.

[Hypolipidemic and antioxidant effects of pyridyl-3-glyoxylic acid]. / O gipolipidemicheskom i antioksidantnom éffekte piridil-3-glioksilovoi kisloty.

Pyridyl-3-glyoxylic acid reduced the blood cholesterol and triglycerides, normalized the lipoprotein spectrum, markedly decreased the intensity of lipid peroxidation, and increased the superoxide dismutase enzyme activity in the experimental rabbits with cholesterol atherosclerosis model. The efficacy of the compound exceeded that of nicotinic acid used as the reference compound.

Ethylene glycol-mediated tubular injury: identification of critical metabolites and injury pathways.

Ethylene glycol (EG) intoxication produces multisystem organ injury, including acute renal failure. Although EG must be metabolized to toxic intermediates to induce organ damage, the specific metabolite(s) responsible and the underlying pathogenic mechanisms remain poorly defined. To explore these issues, isolated mouse proximal tubular segments (PTSs) were incubated with either varying doses of EG or its prime metabolites (glycolate, glycoaldehyde, glyoxylate, or oxalate for 15 to 60 minutes). Injury was assessed by the percentage of lactate dehydrogenase (LDH) release, LDH destruction, adenosine triphosphate (ATP) depletion, or membrane phospholipid degradation. Toxicities were also assessed in cultured HK-2 cells over 18 hours (by MTT assay). EG, glycolate, and oxalate did not induce overt PTS injury. Conversely, glyoxylate and glycoaldehyde were highly toxic, causing profound ATP depletion and LDH release. Glycoaldehyde also caused enzyme (LDH) and selected phospholipid degradation (phosphatidylethanolamine, phosphatidylserine). These changes were not seen with glyoxylate treatment. Acidosis (pH 6.8) and glycine (2 mmol/L) each blocked glyoxylate, but not glycoaldehyde toxicity, indicating differing injury pathways. Only glycoaldehyde and glyoxylate induced marked HK-2 cell death. We conclude that glycoaldehyde and glyoxylate are the principal metabolites responsible for EG nephrotoxicity and do so by causing ATP depletion and phospholipid and enzyme destruction. Glycine and acidosis, by-products of EG metabolism, can attenuate glyoxylate-mediated injury. This suggests that naturally occurring but incomplete protective pathways may be operative during the evolution of EG cytotoxicity.

Toxicity of the styrene metabolite, phenylglyoxylic acid, in rats after three months' oral dosing.

Male Wistar rats were dosed with 0, 1250, 3750 or 5000 mg/l of phenylglyoxylic acid (PGA) (CAS no. 611-73-4) in the drinking water ad libitum for 3 months. During the entire treatment period, there were no gross signs of toxicity related to PGA. No changes in neurobehavior were found after using a functional observational battery or radial arm maze. An increased relative kidney weight was seen in the highest dose-group (Controls: 0.504 +/- 0.031 g/100 g b.wt.; 5000 mg PGA/l: 0.579 +/- 0.033 g/100 g b.wt.; p<0.01). No other organ weights were affected. Histopathology revealed no change in kidney structure. No changes in clinical biochemistry. In the highest dose-group three animals out of ten showed reduction in peripheral nerve myelin sheath thickness. No such changes were seen in the control group. The study revealed no changes in auditory brain stem response but minor changes in electroretinography. The noradrenaline (NA) concentration decreased in pons and thalamus whereas it increased in medulla oblongata and whole brain. The dopamine (DA) concentration increased in cerebellum, hippocampus, pons, and whole brain. The most marked DA increase was seen in hippocampus (Controls: 0.56 +/- 0.10 nmol/g tissue; 5000 mg/l: 1.04 +/- 0.11 nmol/g tissue; p<0.001). The 5-hydroxytryptamine (5-HT) concentration decreased in cerebellum, cerebral cortex, hippocampus, and medulla oblongata, whereas it increased in thalamus. The yield of synaptosomal protein, synaptosomal NA, DA, and 5-HT concentrations, and DA uptake rate were not affected. When dosed males were mated with naive females, there were no differences between groups in the pregnancy rate, number of corpora luteae, implantations, live or dead fetuses, resorptions, preimplantation loss, or postimplantation loss. It is concluded that a part of the effects on kidney, peripheral nerves, and vision, which have previously been reported after exposure to styrene, might be induced by the styrene metabolite, PGA. If PGA has ototoxic effects in rats, the dosing in the present study is not sufficient to induce the necessary ototoxic concentration in blood. Alternatively, the ototoxicity of styrene, like toluene, may be caused the parent compound itself and not by a metabolite like PGA.

[The effect of the oxalate precursors on experimental calcium oxalate urolithogenesis in rats: acute and chronic administration].

Glycolate and glyoxylate, documented lithogenic precursors of oxalate, were administered acutely and chronically to Wistar-strain rats in order to study their effects on oxalate excretion and subsequent stone formation. Urinary oxalate increased significantly within 4 hours, with a maximum being reached between 4-8 hours after a single administration of glycolic acid (200 mg) or glyoxylic acid (200mg). The 24-hour increase in urinary oxalate was about 3% of each amount given. Hyperoxaluria developed immediately and persisted throughout the experimental period in all the rats, which were fed on a diet containing glycolic acid or glyoxylic acid at a 3% level. Microscopically amorphous substances accumulated in the renal tubules at one week. Significant crystal formation appeared in the tubules after two weeks in both experimental groups and consistently increased both in number and in volume until the 4th week. Therefore, the oral administration of either glycolate or glyoxylate increased urinary oxalate comparably much within a few hours, but a few weeks of hyperoxaluria may be necessary to develop crystals in the convoluted tubules.

A "chemical" concept for the therapy of glyoxylate-induced oxalurias (1).

Glyoxylic acid is the toxic principle of acquired and inherited oxalurias. A "chemical", not enzyme-mediated detoxication concept for the trapping of this aldehyde is described, based on a spontaneous formation of alkaloid-type heterocycles by reaction with biogenic amines or amino acids. 5,5-Dimethylthiazolidine-2(R,S)-4(S)-dicarboxylic acid, prepared by the condensation of D(-)-penicillamine with glyoxylic acid, was found to be formed quickly in vitro, to be stable in vivo and of good physiological compatibility. Renal elimination of the unchanged thiazolidine occurs mainly within 24 h, after administration of its calcium salt to NMRI-mice. Recovery up to 85% of the applied dose was quantitatively monitored by HPLC after derivatization to the corresponding fluorescent dansyl compound, which was unequivocally identified by MS analysis after isolation from mice urine.