Visualisation of calcium oxalate crystal macropatterns in plant leaves using an improved fast preparation method.

Leaves of the majority of plants contain calcium oxalate (CaOx) crystals or druses which often occur in spectacular distribution patterns. Numerous studies on CaOx in plant tissues across many different plant groups have been published, since it can be visualised readily under a light microscope (LM). However, there is surprisingly limited knowledge on the actual, precise distribution of CaOx in the leaves of quite ordinary plants such as common native and exotic trees. Traditional sample preparation for the documentation of the distribution of CaOx crystals in a given sample - including overall distribution - requires time-consuming clearing procedures. Here we present a refined fast preparation method to visualise the overall CaOx complement in a sample: The plant material is ashed and the ash viewed under the polarising microscope. This is a rapid method which overcomes many shortcomings of other methods and permits the visualisation of the entire CaOx content in most leaf samples. Pros and cons in comparison with the conventional clearing technique are discussed. Further aspects for CaOx investigations by micro-CT and scanning electron microscopy are discussed.

Multi-element imaging of urinary stones by LA-ICP-MS with a homogeneous co-precipitation CaC2O4-matrix calibration standard.

Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) studies on trace element concentration and their spatial distribution in CaC2O4-matrix urinary stones are important but powerfully rely on matrix-matched external calibration. In this work, CaC2O4 precipitate CaOx-1 which was doped with Mg, Cr, Mn, Fe, Co, Cu, Zn, and Sr was prepared by the homogeneous co-precipitation method. It had a homogeneous distribution of major (RSD of 0.46%) and trace elements (RSD of 1.83-6.92%) due to the negligible concentration difference compared with that prepared by the heterogeneous co-precipitation method. Based on this, an analytical method for quantitative determination of elemental concentration in CaC2O4-matrix samples was established using CaOx-1 as a calibration standard, and the accuracy of this method was assessed by calibrating the elemental concentration in another synthetic CaC2O4 precipitate CaOx-2 with relative deviation (Dr) from - 11.43% (Mn) to 9.76% (Mg). Finally, a methodology for quantitative imaging of Mg, Cr, Mn, Fe, Co, Cu, Zn, and Sr in urinary stones via LA-ICP-MS was developed. From the elemental distributional maps, an annular texture can be found for Mg, Cu, Zn, and Sr, which corresponds to the annular white and brown texture in the real urinary stone. A homogeneous distribution of Fe and low concentrations of Cr and Co were found throughout the stone, while Mn was highly concentrated in the margin of the stone. All these results demonstrate that quantitative distribution patterns of Mg, Cr, Mn, Fe, Co, Cu, Zn, and Sr can be obtained by LA-ICP-MS using CaOx-1 as a calibration standard, which can provide potential evidence for urological and other medical studies.

Composition of Urine Collected from Non-Stone-Forming Chinese Persons during Different Short-Term Periods of the Day.

The purpose of this study in a small group of non-stone-forming Chinese persons was to measure the levels of supersaturation with calcium oxalate and calcium phosphate and pH with the aim of confirming if any of the different short-term urine samples were better for risk evaluation than a 24-h sample. Nine normal men and 1 woman collected urine during 4 periods of the day. Period 1 between 08 and 12 h, Period 2 between 12 and 18 h, Period 3 between 18 and 22 h, and Period 4 between 22 and 08 h. Each sample was analysed for calcium, oxalate, citrate, magnesium and phosphate, and estimates of supersaturation with calcium oxalate (CaOx) and calcium phosphate (CaP) were expressed in terms of AP(CaOx) and AP(CaP) index. An estimate of the solute load of CaOx was also calculated. Urine composition for 24-h urine (Period 24) was obtained mathematically from the analysed variables. Urine composition corresponding to 14-h urine portions 22-12 h (Period 14N) and 08-22 h (Period 14 D) were calculated. The lowest pH levels were recorded in Period 1 urine. The highest level of AP(CaOx) index was recorded during Period 1, and the product AP(CaOx) index × 107 × hydrogen ion concentration was significantly higher in Period 1 urine than in 24-h urine (p = 0.02). Also, the product SL(CaOx) × 107 × hydrogen ion concentration was significantly higher in Period 1 urine (p = 0.02). Low AP (CaP) index levels were recorded in Period 4, but also in all periods following dietary loads of calcium and phosphate. With the important reservation that the analytical results were obtained from non-stone-forming persons, the conclusion is that analysis of urine samples collected between 08 and 12 h might be an alternative to 24-h urine. The risk evaluation might advantageously be expressed either in terms of the product AP(CaOx) index × 107 × hydrogen ion concentration or the product SL(CaOx) × 107 × hydrogen ion concentration.

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.

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.

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.

Effect of sample time on urinary lithogenic risk indexes in healthy and stone-forming adults and children.

BACKGROUND: The diagnosis and follow-up of stone forming patients is usually performed by analysis of 24-h urine samples. However, crystallization risk varies throughout the day, being higher at night. The main objective of this study is to evaluate the urinary crystallization risk in adults and children by calculating risk indexes based on different collection periods. METHODS: The study included 149 adults (82 healthy and 67 stone-formers) and 108 children (87 healthy and 21 stone-formers). 24-h urine was collected, divided into 12-h daytime sample (8 am to 8 pm), and 12-h overnight sample (8 pm to 8 am next morning). Solute concentrations, the calcium to citrate ratio (Ca/Cit), and the ion activity product of calcium oxalate (AP[CaOx]) and calcium phosphate (AP[CaP]) were calculated in each 12-h sample and in overall 24-h urine. Assessments were also related to stone type. RESULTS: Ca/Cit and AP(CaOx) were significantly higher in stone forming patients than in healthy subjects. The 12-h overnight samples had the highest values for both risk indexes, confirming a greater risk for crystallization at night. The AP(CaP) index was significantly higher in patients with pure hydroxyapatite stones than healthy controls, but was not significantly different between stone-formers overall and healthy controls. CONCLUSIONS: The calculation of risk indexes is a simple method that clinicians can use to estimate crystallization risk. For this purpose, the use of 12-h overnight urine may be a reliable alternative to 24-h collections.

Regulation of bone mineral density in the grey squirrel, Sciurus carolinensis: Bioavailability of calcium oxalate, and implications for bark stripping.

The damage caused when grey squirrels strip the outer bark off trees and ingest the underlying phloem can result in reduced timber quality or tree death. This is extremely costly to the UK forestry industry and can alter woodland composition, hampering conservation efforts. The calcium hypothesis proposes that grey squirrels ingest phloem to ameliorate a seasonal calcium deficiency. Calcium in the phloem predominantly takes the form of calcium oxalate (CaOx), however not all mammals can utilise CaOx as a source of calcium. Here, we present the results of a small-scale study to determine the extent to which grey squirrels can utilise CaOx. One of three custom-made diets containing calcium in varying forms and quantities (CaOx diet, Low-calcium carbonate (CaCO3 ) diet and Control diet) were fed to three treatment groups of six squirrels for 8 weeks. Bone densitometric properties were measured at the end of this time using peripheral quantitative computed tomography and micro-computed tomography. Pyridinoline-a serum marker of bone resorption-was measured regularly throughout the study. Bone mineral density and cortical mineralisation were lower in squirrels fed the CaOx diet compared to the Control group but similar to that of those on the Low-calcium diet, suggesting that calcium from calcium oxalate was not effectively utilised to maintain bone mineralisation. Whilst no differences were observed in serum pyridinoline levels between individuals on different diets, females had on average higher levels than males throughout the study. Future work should seek to determine if this apparent lack of ability to utilise CaOx is common to a large sample of grey squirrels and if so, whether it is consistent across all areas and seasons.

Gomphrena claussenii, a novel metal-hypertolerant bioindicator species, sequesters cadmium, but not zinc, in vacuolar oxalate crystals.

Gomphrena claussenii is a recently described zinc (Zn)- and cadmium (Cd)-hypertolerant Amaranthaceae species displaying a metal bioindicator Zn/Cd accumulation response. We investigated the Zn and Cd distribution in stem and leaf tissues of G. claussenii at the cellular level, and determined metabolite profiles to investigate metabolite involvement in Zn and Cd sequestration. Gomphrena claussenii plants exposed to high Zn and Cd supply were analysed by scanning electron microscopy with energy-dispersive X-ray (SEM-EDX) and micro-proton-induced X-ray emission (micro-PIXE). In addition, gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) was used to determine metabolite profiles on high Zn and Cd exposure. Stem and leaf tissues of G. claussenii plants exposed to control and high Cd conditions showed the abundant presence of calcium oxalate (CaOx) crystals, but on high Zn exposure, their abundance was strongly reduced. Ca and Cd co-localized to the CaOx crystals in Cd-exposed plants. Citrate, malate and oxalate levels were all higher in shoot tissues of metal-exposed plants, with oxalate levels induced 2.6-fold on Zn exposure and 6.4-fold on Cd exposure. Sequestration of Cd in vacuolar CaOx crystals of G. claussenii is found to be a novel mechanism to deal with Cd accumulation and tolerance.

In Situ Accumulation of CaOx Crystals in C. quitensis Leaves and Its Relationship with Anatomy and Gas Exchange.

The accumulation of crystal calcium oxalate (CaOx) in plants is linked to a type of stress-induced photosynthesis termed 'alarm photosynthesis', serving as a carbon reservoir when carbon dioxide (CO2) exchange is constrained. Colobanthus quitensis is an extremophyte found from southern Mexico to Antarctica, which thrives in high-altitude Andean regions. Growing under common garden conditions, C. quitensis from different latitudinal provenances display significant variations in CaOx crystal accumulation. This raises the following questions: are these differences maintained under natural conditions? And is the CaOx accumulation related to mesophyll conductance (gm) and net photosynthesis (AN) performed in situ? It is hypothesized that in provenances with lower gm, C. quitensis will exhibit an increase in the use of CaOx crystals, resulting in reduced crystal leaf abundance. Plants from Central Chile (33°), Patagonia (51°), and Antarctica (62°) were measured in situ and sampled to determine gas exchange and CaOx crystal accumulation, respectively. Both AN and gm decrease towards higher latitudes, correlating with increases in leaf mass area and leaf density. The crystal accumulation decreases at higher latitudes, correlating positively with AN and gm. Thus, in provenances where environmental conditions induce more xeric traits, the CO2 availability for photosynthesis decreases, making the activation of alarm photosynthesis feasible as an internal source of CO2.

The modulatory effects of large and small extracellular vesicles from normal human urine on calcium oxalate crystallization, growth, aggregation, adhesion on renal cells, and invasion through extracellular matrix: An in vitro study.

Urinary extracellular vesicles (uEVs) play important roles in physiologic condition and various renal/urological disorders. However, their roles in kidney stone disease remain unclear. This study aimed to examine modulatory effects of large and small uEVs derived from normal human urine on calcium oxalate (CaOx) crystals (the main component in kidney stones). After isolation, large uEVs, small uEVs and total urinary proteins (TUPs) with equal (protein equivalent) concentration were added into various crystal assays to compare with the control (without uEVs or TUPs). TUPs strongly inhibited CaOx crystallization, growth, aggregation and crystal-cell adhesion. Large uEVs had lesser degree of inhibition against crystallization, growth and crystal-cell adhesion, and comparable degree of aggregation inhibition compared with TUPs. Small uEVs had comparable inhibitory effects as of TUPs for all these crystal assays. However, TUPs and large uEVs slightly promoted CaOx invasion through extracellular matrix, whereas small uEVs did not affect this. Matching of the proteins reported in six uEVs datasets with those in the kidney stone modulator (StoneMod) database revealed that uEVs contained 18 known CaOx stone modulators (mainly inhibitors). These findings suggest that uEVs derived from normal human urine serve as CaOx stone inhibitors to prevent healthy individuals from kidney stone formation.

The direct inhibitory effects of Lactobacillus acidophilus, a commensal urinary bacterium, on calcium oxalate stone development.

BACKGROUND: Lactobacillus acidophilus is a commensal urinary bacterium found more abundantly in healthy individuals than in stone patients. Hence, it has been proposed to play an inhibitory role in kidney stone disease (KSD) but with unclear mechanisms. We therefore investigated the direct effects of L. acidophilus on calcium oxalate (CaOx) stone development compared with Escherichia coli, which is known to promote CaOx stone formation. RESULTS: L. acidophilus at 1 × 103 CFU/ml  significantly reduced the abundance of newly formed crystals, enlargement and aggregation of seeded crystals, and crystal adhesion on renal cell membranes. By contrast, E. coli at 1 × 103 CFU/ml significantly enhanced crystal growth and aggregation but did not affect crystallization and crystal-cell adhesion. Oxalate consumption assay showed that neither L. acidophilus nor E. coli significantly reduced the remaining oxalate level after 1 - 3 h incubation. However, both of them adhered to CaOx crystals. Surface component detection revealed that only L. acidophilus expressed S-layer protein, whereas only E. coli exhibited flagella on their surfaces. Removal of L. acidophilus S-layer protein and E. coli flagella completely abolished the inhibitory and promoting effects of L. acidophilus and E. coli, respectively. CONCLUSIONS: L. acidophilus inhibits CaOx stone development by hampering crystallization, growth, aggregation and cell-adhesive ability of CaOx. By contrast, E. coli enhances CaOx stone development by promoting CaOx growth and aggregation. Their contradictory effects are most likely from differential surface components (i.e., S-layer protein on L. acidophilus and flagella on E. coli) not from oxalate-degrading ability. Video Abstract.

Optimizing 'red Fuji' apple quality: Auxin-mediated calcium distribution via fruit-stalk in bagging practices.

In apples, a bottleneck effect in calcium (Ca) transport within fruit stalk has been observed. To elucidate that how auxin affects Ca forms and distribution in the apple fruit stalk, we investigated the effects of different concentrations of auxin treatment (0, 10, 20, and 30 mg·L-1) on Ca content, forms, distribution, and fruit quality during later stages of fruit expansion. The results showed that auxin treatment led to a dramatic reduction in total Ca content in stalk, while an approximately 30 % increase in fruit. Furthermore, auxin treatment effectively enhanced the functionality of xylem vessels in vascular bundles of the stalk in bagged apples. Finally, TOPSIS method was used to assess fruit quality, with treatments ranked as follows: IAA20 > NAA20 > IAA30 > IAA10 > CK > NPA. The findings lay a foundation for further studies on the bottleneck in Ca transport within stalk, uneven distribution of Ca in fruit, and provide insights into Ca utilization efficiency in bagged apples.

Research on key pathogenesis and potential intervention targets of idiopathic renal calculi composed of calcium oxalate (CaOx) based on bioinformatics.

Background: Calcium oxalate (CaOx) kidney stones are the most common type of stones in the urinary system, and their formation involves a complex mechanism with multiple contributing factors. In recent years, with the development of bioinformatics, there has been a deeper understanding of the pathogenesis of this type of disease. This study aimed to analyze the gene expression profiles of idiopathic kidney stones composed of CaOx using bioinformatics methods. By investigating the pathogenesis at the molecular level and identifying potential therapeutic targets, the study also integrated clinical data to validate the clinical relevance of the target genes. Methods: Gene expression profiles from the GSE73680 dataset were analyzed via the Gene Expression Omnibus (GEO) database to identify differentially expressed genes (DEGs) between Randall's plaques (RPs) from kidney papillae associated with CaOx stones and normal kidney papillae tissues. The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database was employed to construct transcription factor (TF)-DEG-microRNA (miRNA) networks, and key genes were screened using the Molecular Complex Detection (MCODE) plugin. A gene set enrichment analysis (GSEA) was performed to investigate the possible underlying mechanisms of the key genes. The clinical data of idiopathic CaOx kidney stone patients who received treatment at the General Hospital of Northern Theater Command from January 2020 to December 2022 were retrospectively analyzed. Enzyme-linked immunosorbent assay (ELISA) kits were used to measure the transcriptional activity of the key genes in calcified kidney papillae tissues. Univariate and multivariate logistic regression analyses were employed to analyze the transcriptional activity of the key genes and their association with idiopathic kidney stones composed of CaOx. Results: In the GSE73680 dataset, 276 upregulated and 538 downregulated DEGs were identified. Protein-protein interaction network construction revealed one significant module and three candidate genes [interleukin 11 (IL-11), interleukin 16 (IL-16), and interleukin 32 (IL-32)]. The TF-DEG-miRNA network indicated that IL-11 might be regulated by 25 TFs and interact with six miRNAs. The GSEA suggested that IL-11 could influence the development of idiopathic CaOx stones through chemokine expression and via the signaling pathways of the nucleotide-binding oligomerization domain-like receptors [NOD-like receptors (NLRs)] and toll-like receptors (TLRs). The clinical data analysis revealed that the IL-11 serum levels were significantly elevated in the patients with idiopathic kidney stones composed of CaOx compared to the control subjects (P<0.001). Additionally, IL-11 was identified as an independent risk factor for the development of idiopathic CaOx kidney stones (P<0.001). Conclusions: The bioinformatically identified key genes and signaling pathways provide a deeper understanding of the potential mechanisms underlying idiopathic CaOx kidney stones. Preliminary clinical trials suggest that elevated serum IL-11 levels in idiopathic CaOx kidney stone patients could serve as a possible diagnostic biomarker and treatment target.

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.

Knockout of the aryl hydrocarbon receptor results in distinct hepatic and renal phenotypes in rats and mice.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor which plays a role in the development of multiple tissues and is activated by a large number of ligands, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In order to examine the roles of the AHR in both normal biological development and response to environmental chemicals, an AHR knockout (AHR-KO) rat model was created and compared with an existing AHR-KO mouse. AHR-KO rats harboring either 2-bp or 29-bp deletion mutation in exon 2 of the AHR were created on the Sprague-Dawley genetic background using zinc-finger nuclease (ZFN) technology. Rats harboring either mutation type lacked expression of AHR protein in the liver. AHR-KO rats were also insensitive to thymic involution, increased hepatic weight and the induction of AHR-responsive genes (Cyp1a1, Cyp1a2, Cyp1b1, Ahrr) following acute exposure to 25 µg/kg TCDD. AHR-KO rats had lower basal expression of transcripts for these genes and also accumulated ~30-45-fold less TCDD in the liver at 7 days post-exposure. In untreated animals, AHR-KO mice, but not AHR-KO rats, had alterations in serum analytes indicative of compromised hepatic function, patent ductus venosus of the liver and persistent hyaloid arteries in the eye. AHR-KO rats, but not AHR-KO mice, displayed pathological alterations to the urinary tract: bilateral renal dilation (hydronephrosis), secondary medullary tubular and uroepithelial degenerative changes and bilateral ureter dilation (hydroureter). The present data indicate that the AHR may play significantly different roles in tissue development and homeostasis and toxicity across rodent species.

NEAT1 Regulates Calcium Oxalate Crystal-Induced Renal Tubular Oxidative Injury via miR-130/IRF1.

Aims: Calcium oxalate (CaOx) crystal deposition induces damage to the renal tubular epithelium, increases epithelial adhesion, and contributes to CaOx nephrocalcinosis. The long noncoding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) is thought to be involved in this process. In this study, we aimed to investigate the mechanism by which NEAT1 regulates renal tubular epithelium in response to inflammatory and oxidative injury triggered by CaOx crystals. Results: As CaOx crystals were deposited in mouse kidney tissue, the expression of NEAT1 was significantly elevated and positively correlated with interferon regulatory factor 1 (IRF1), Toll-like receptor 4 (TLR4), and NF-κB. NEAT1 targets and inhibits miR-130a-3p as a competitor to endogenous RNA. miR-130 binds to and exerts inhibitory effects on the 3'-untranslated region of IRF1. After transfected with silence-NEAT1, IRF1, TLR4, and NF-κB were also variously inhibited, and oxidative damage in renal calcinosis was subsequently attenuated. When we simultaneously inhibited NEAT1 and miR-130, renal tubular injury was exacerbated. Innovation and Conclusion: We found that the lncRNA NEAT1 can enhance IRF1 signaling through targeted repression of miR-130a-3p and activate TLR4/NF-κB pathways to promote oxidative damage during CaOx crystal deposition. This provides an explanation for the tubular epithelial damage caused by CaOx crystals and offers new ideas and drug targets for the prevention and treatment of CaOx nephrocalcinosis. Antioxid. Redox Signal. 38, 731-746.

p53 deacetylation alleviates calcium oxalate deposition-induced renal fibrosis by inhibiting ferroptosis.

Calcium oxalate (CaOx) stones are among the most common types of kidney stones and are associated with renal tubular damage, interstitial fibrosis, and chronic kidney disease. The mechanism of CaOx crystal-induced renal fibrosis remains unknown. Ferroptosis, a type of regulated cell death, is characterised by iron-dependent lipid peroxidation, and the tumour suppressor p53 is a key regulator of ferroptosis. In the present study, our results demonstrated that ferroptosis was significantly activated in patients with nephrolithiasis and hyperoxaluric mice as well as verified the protective effects of ferroptosis inhibition on CaOx crystal-induced renal fibrosis. Moreover, the single-cell sequencing database, RNA-sequencing, and western blot analysis revealed that the expression of p53 was increased in patients with chronic kidney disease and the oxalate-stimulated human renal tubular epithelial cell line, HK-2. Additionally, the acetylation of p53 was enhanced by oxalate stimulation in HK-2 cells. Mechanistically, we found that the induction of p53 deacetylation, owing to either the SRT1720-induced activation of deacetylase sirtuin 1 or the triple mutation of p53, inhibited ferroptosis and alleviated renal fibrosis caused by CaOx crystals. We conclude that ferroptosis is one of the critical mechanisms contributing to CaOx crystal-induced renal fibrosis, and the pharmacological induction of ferroptosis via sirtuin 1-mediated p53 deacetylation may be a potential target for preventing renal fibrosis in patients with nephrolithiasis.

Overexpression of sirtuin 1 attenuates calcium oxalate-induced kidney injury by promoting macrophage polarization.

Sirtuin 1 (SIRT1) protein is involved in macrophage differentiation, while NOTCH signaling affects inflammation and macrophage polarization. Inflammation and macrophage infiltration are typical processes that accompany kidney stone formation. However, the role and mechanism of SIRT1 in renal tubular epithelial cell injury caused by calcium oxalate (CaOx) deposition and the relationship between SIRT1 and the NOTCH signaling pathway in this urological disorder are unclear. This study investigated whether SIRT1 promotes macrophage polarization to inhibit CaOx crystal deposition and reduce renal tubular epithelial cell injury. Public single-cell sequencing data, RT-qPCR, immunostaining approaches, and Western blotting showed decreased SIRT1 expression in macrophages treated with CaOx or exposed to kidney stones. Macrophages overexpressing SIRT1 differentiated towards the anti-inflammatory M2 phenotype, significantly inhibiting apoptosis and alleviating injury in the kidneys of mice with hyperoxaluria. Conversely, decreased SIRT1 expression in CaOx-treated macrophages triggered Notch signaling pathway activation, promoting macrophage polarization towards the pro-inflammatory M1 phenotype. Our results suggest that SIRT1 promotes macrophage polarization towards the M2 phenotype by repressing the NOTCH signaling pathway, which reduces CaOx crystal deposition, apoptosis, and damage in the kidney. Therefore, we propose SIRT1 as a potential target for preventing disease progression in patients with kidney stones.

A highlightedly improved method for isolating and characterizing calcium oxalate crystals from tubercles of Mammillaria schumannii.

BACKGROUND: Calcium oxalate (CaOx) is the most prevalent and widespread biomineral in plants and is involved in protective and/or defensive functions against abiotic stress factors. It is, however, expected that this function has an extremely significant contribution to growth processes in plants bearing large amounts of CaOx, such as cacti growing in desert environment. RESULTS: In our research, small-sized CaOx crystals (≤ 20 µm) with tetrahedral or spherical shapes were observed to dominate in each epidermal and cortical cell from the tubercles of Mammillaria schumannii, a species from the Cereoideae subfamily, having tubercles (main photosynthetic organs) united with adjacent ones almost into ridges on its stem. Because they have potential significant functions, differential centrifugations after mechanical blending were used to obtain these small-sized CaOx crystals, which extremely tend to adhere to tissue or suspend in solution. And then the combined Scanning Electron Microscope Energy Dispersive System (SEM-EDS) and Raman spectroscopy were further performed to demonstrate that the extracted crystals were mainly CaC2O4·2H2O. Interestingly, spherical druses had 2 obvious abnormal Raman spectroscopy peaks of -CH and -OH at 2947 and 3290 cm-1, respectively, which may be attributed to the occluded organic matrix. The organic matrix was further extracted from spherical crystals, which could be polysaccharide, flavone, or lipid compounds on the basis of Raman spectroscopy bands at 2650, 2720, 2770, and 2958 cm-1. CONCLUSIONS: Here we used a highlightedly improved method to effectively isolate small-sized CaOx crystals dominating in the epidermal and cortical cells from tubercles of Mammillaria schumannii, which extremely tended to adhere plant tissues or suspend in isolation solution. And then we further clarified the organic matrix getting involved in the formation of CaOx crystals. This improved method for isolating and characterizing biomineral crystals can be helpful to understand how CaOx crystals in cacti function against harsh environments such as strong light, high and cold temperature, and aridity.