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Calcium oxalate (CaOx) nephrolithiasis constitutes approximately 75% of nephrolithiasis cases, resulting from the supersaturation and deposition of CaOx crystals in renal tissues. Despite their prevalence, precise biomarkers for CaOx nephrolithiasis are lacking. With advances in high-throughput sequencing, we aimed to identify biomarkers of CaOx nephrolithiasis by combining two CaOx nephrolithiasis datasets (GSE73680 and GSE117518). Utilizing weighted gene co-expression network analysis (WGCNA) and four machine learning, we identified six hub genes (DLK2, BHLHA15, C12orf5, ICMT, LOXHD1, and TP73) as potential biomarkers. Additionally, CIBERSORT immune infiltration analysis suggested that these core genes may influence immune cell recruitment and infiltration in CaOx nephrolithiasis. Then, TP73 emerged as a significant hub gene in CaOx nephrolithiasis via receiver operating characteristic (ROC) analysis (AUC = 0.885). Furthermore, the role of TP73 was validated in CaOx nephrolithiasis rat models induced by 1% ethylene glycol, as well as clinical samples and renal tubular epithelial cell models treated with 1 mM oxalate. Immunohistochemistry, RNA-Sequencing, and RT-qPCR experiments demonstrated an increased expression of TP73 in CaOx nephrolithiasis rat models and clinical samples. After transfection with TP73 lentivirus, CCK-8 assays suggested that TP73 could inhibit the proliferation of HK-2 and NRK-52E cells. In oxalate-induced cell models, dihydroethidium staining and flow cytometry apoptosis assays indicated that TP73 could enhance ROS levels and cell apoptosis. In summary, our study preliminarily identified TP73 as a diagnostic biomarker and elucidated the promoting role of TP73 in CaOx nephrolithiasis, providing a deeper understanding of the clinical diagnosis and pathogenesis., Competing Interests: Declarations Conflict of interests The authors declare no competing interests. Ethical approval This study was approved by the Fudan Laboratory Animal Ethics Board and the Huashan Institutional Review Board of Fudan University (HIRB). Consent for publication Not applicable., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Our comprehensive genomic investigation employing tree shrew calcium oxalate stone models unveils intricate links between kidney stone formation and diverse physiological systems. We identify a constellation of genes whose expression patterns point to multifaceted interactions among cardiovascular health, renal fibrosis, and bone homeostasis in the pathogenesis of renal calculi. Key players include CHIT1, TNFRSF18, CLEC4E, RGS1, DCSTAMP, and SLC37A2, which emerge as pivotal actors in arteriosclerosis, renal fibrosis, and osteoclastogenesis respectively, showcasing the complexity of stone disease. The downregulation of ADRA1D, LVRN, and ABCG8 underscores roles in urodynamics, epithelial-mesenchymal transition, and vitamin D metabolism, linking these to nephrolithiasis. Comparative genomics across tree shrew, human (Randall's plaque), rat, and mouse identifies shared KEGG pathways including Calcium signaling, Actin cytoskeleton regulation, Neuroactive ligand-receptor interactions, Complement and coagulation cascades, TRP channel regulation by inflammatory mediators, p53 signaling, and Fc gamma R-mediated phagocytosis. These pathways underscore the interconnectedness of immune, inflammatory, and metabolic processes in stone development. Our findings suggest novel targets for future therapeutics and prevention strategies against nephrolithiasis, highlighting the need for a holistic view of the disease encompassing multiple pathogenic factors., Competing Interests: Declarations Ethical approval This study was reviewed and approved by the Animal Experiment Ethics Committee of Kunming Medical University (Approval no: kmmu20230176). Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Kidney stones result from abnormal biomineralization, although the mechanism behind their formation remains unclear. Annexin A6 (AnxA6), a calcium-dependent lipid-binding protein, is associated with several mineralization-related diseases, but its role in kidney stones is unknown. This study aimed to explore the role and mechanism of AnxA6 in calcium oxalate (CaOx) kidney stones. An in vitro model in which renal tubular epithelial cells (RTECs) were treated with 1 mmol/L oxalate was established, and AnxA6 protein and mRNA expression were examined. Genetic engineering, drug intervention, and biochemical assays were used to investigate the role of AnxA6. The results revealed that AnxA6 was significantly overexpressed in the CaOx model. AnxA6 knockdown in RTECs reduced oxalate-induced oxidative stress, ROS accumulation, and mitochondrial damage, whereas AnxA6 overexpression exacerbated these effects. Blocking ryanodine receptor-mediated calcium release reversed AnxA6-induced oxidative damage. Additionally, AnxA6 increased oxalate adhesion to RTECs by binding to oxalate. In conclusion, AnxA6 contributes to CaOx kidney stone formation by promoting both oxidative stress via calcium release and crystal-cell adhesion by binding to oxalate. This study offers new insight into CaOx kidney stone formation., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objectives: To explore the role of S100A9 protein in renal calcium oxalate (CaOx) stone formation., Methods: CaOx nephrocalcinosis mice were established via intraperitoneal injection of glyoxylate. They were treated with S100A9 deficiency, Paquinimod, or p38 MAPK-IN-1. Vonkossa staining was conducted to observe the deposition of CaOx crystals. Renal expression of inflammation, macrophage polarization, and injury markers was detected using immunohistochemistry and qPCR. Effects of S100A9 on renal tubular epithelial cells (HK-2) were explored by transcriptome sequencing. The mechanism of how S100A9 regulated lipocalin 2 (LCN2) was studied through Western Blot. Flow cytometry was performed to detect the influence of LCN2 on macrophages polarization., Results: S100A9 deficiency inhibited the renal deposition of CaOx crystals in nephrocalcinosis mice. S100A9 upregulated the expression of LCN2 in HK-2 cells via activating the TLR4-p38/MAPK pathway. LCN2 promoted the migration and M1 polarization of macrophages. S100A9 deficiency downregulated the renal expression of LCN2, IL1-β, Kim-1, F4/80, and CD80 in nephrocalcinosis mice. Paquinimod and p38 MAPK-IN-1 both inhibited the renal deposition of CaOx crystals and downregulated the expression of LCN2, IL1-β, Kim-1, F4/80, iNOS, and CD68 in nephrocalcinosis mice., Conclusions: S100A9 promotes renal inflammatory injury by activating the TLR4-p38/MAPK-LCN2 pathway and then contributes to CaOx stone formation., Competing Interests: Declaration of competing interest Wang Qing reports financial support was provided by National Natural Science Foundation of China. Wang Qing reports financial support was provided by The Guizhou Provincial Science and Technology Project. Wang Qing reports financial support was provided by Subsidy funding for the 2021 National Natural Science Foundation of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The mitochondrion serves as a critical intracellular organelle, engaging in essential roles in the regulation of energy production, oxidative stress management, calcium homeostasis, and apoptosis. One such disease that has been particularly associated with these functions is kidney stone disease (KSD), specifically calcium oxalate (CaOx). It is underpinned by oxidative stress and tissue inflammation. Recent studies have shed light on the vital involvement of mitochondrial dysfunction, the nucleotide-binding domain and leucine-rich repeat containing protein 3 (NLRP3) inflammasome, endoplasmic reticulum stress and subsequent cell death in CaOx crystal retention and aggregation. These processes are pivotal in the pathogenesis of kidney stone formation. This review focuses on the pivotal roles of mitochondria in renal cell functions and provides an overview of the intricate interconnectedness between mitochondrial dysfunction and NLRP3 inflammasome activation in the context of KSD. It is essential to recognise the utmost significance of gaining a comprehensive understanding of the mechanisms that safeguard mitochondrial function and regulate the NLRP3 inflammasome. Such knowledge carries significant scientific implications and opens up promising avenues for the development of innovative strategies to prevent the formation of kidney stones., (© 2024 BJU International.)

As typical examples of pathological biomineralization, urinary stones and stent encrustation have been associated with bacteria, yet the underlying mechanisms remain unclear. In this study, the effect of Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus on the nucleation and growth of calcium oxalate crystals both in solution and on material surfaces in vitro was investigated. Both bacteria can promote calcium oxalate crystallization, and E. coli shows a prominent ability to boost the nucleation and growth rate. Interestingly, we discovered an Ostwald ripening phenomenon after the initial nucleation on the material surfaces, where larger particles emerge upon the disappearance of small nuclei particles, evident in the case of S. aureas . Over an extended period of time, erosion and disintegration of the crystals was observed when bacteria were involved. Based on these understandings, we developed a new functional surface by synthesizing an antibacterial polypeptoid in-house and utilizing polyurethane as the substrate material. This surface exhibits a synergistic effect that inhibits the formation of calcium oxalate crystals. This study helps to elucidate the role of bacteria in calcium oxalate biomineralization and supports further development of treatment approaches such as anti-encrustation polymer materials.

The oxalate-carbonate pathway (OCP) involves degradation of soil oxalate to carbonate. To exploit and manage this natural mineralization of assimilated atmospheric CO 2 into stable carbonates, improved understanding of this complex biotransformation process is needed. A strain of oxalate-degrading bacteria, Azospirillum sp. OX-1, was isolated from soil, and its secondary products of calcium oxalate degradation were analyzed and characterized using SEM, XRD, TG/DTG-DTA and FTIR-spectroscopy. The molecular mechanism of calcium oxalate degradation was also analyzed using proteomics. The results showed, for the first time, that OX-1 could not only degrade calcium oxalate to calcium carbonate, but also that the process was accompanied by synthesis of methane. Proteomic analysis demonstrated that OX-1 has a dual enzyme system for calcium oxalate degradation, using formyl-CoA transferase (FRC) and thiamine pyrophosphate (ThDP)-dependent oxalyl-CoA decarboxylase (OXC) to form calcium carbonate. Up-regulated expression of enzymes related to methane synthesis was also detected during calcium oxalate degradation. Since methane is also a potent greenhouse gas, these new results suggest that the utility of exploiting the OCP to reduce atmospheric CO 2 must be re-evaluated and that further studies should be conducted to reveal how widespread the methane producing capacity of strain OX-1 is in other bacteria and soil environments., (© 2024. The Author(s).)

Puerarin(PUE), an isoflavonoid extracted from Pueraria root, has anti-apoptotic effects. The objective of this research is to examine the impact of PUE on renal apoptosis and inflammation resulting from renal calculi and to elucidate its mechanism. The approach of network pharmacology and molecular docking was employed to discover potential targets and pathways of PUE. An animal model of calcium oxalate crystal deposition by intraperitoneal injection of glyoxylate and a model of COM-induced human renal tubular epithelial cells (HK2) were used to investigate the pharmacological mechanisms of PUE against apoptosis and inflammation. We used haematoxylin-eosin (H&E) and Periodic Acid-Schiff staining (PAS) to assess the effect of PUE on crystal deposition and damage. The mechanism of PUE was elucidated and validated using Western blotting, histology and immunohistochemical staining. Network pharmacology findings indicated that the PI3K/AKT pathway plays a crucial role in PUE. We experimentally demonstrate that PUE alleviated COM-induced changes in apoptotic proteins, increased inflammatory indicators and changes in oxidative stress-related indicators in HK2 cells by activating the PI3K/AKT pathway, reduced serum creatinine and urea nitrogen levels in mice caused by CaOx, alleviated crystal deposition and damage, and alleviated apoptosis, oxidative stress and inflammation. Puerarin attenuates renal apoptosis and inflammation caused by kidney stones through the PI3K/AKT pathway., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Calcium oxalate (CaOx) kidney stones are common and recurrent, lacking pharmacological prevention. Randall's plaques (RPs), calcium deposits in renal papillae, serve as niduses for some CaOx stones. This study explores the role of osteogenic-like cells in RP formation resembling ossification. CaP crystals deposit around renal tubules, interstitium, and blood vessels in RP tissues. Human renal interstitial fibroblasts (hRIFs) exhibit the highest osteogenic-like differentiation potential compared to chloride voltage-gated channel Ka positive tubular epithelial cells, aquaporin 2 positive collecting duct cells, and vascular endothelial cells, echoing the upregulated osteogenic markers primarily in hRIFs within RP tissues. Utilizing RNA-seq, osteomodulin (OMD) is found to be upregulated in hRIFs within RP tissues and hRIFs following osteogenic induction. Furthermore, OMD colocalizes with CaP crystals and calcium vesicles within RP tissues. OMD can enhance osteogenic-like differentiation of hRIFs in vitro and in vivo. Additionally, crystal deposits are attenuated in mice with Omd deletion in renal interstitial fibroblasts following CaOx nephrocalcinosis induction. Mechanically, a positive feedback loop of OMD/BMP2/BMPR1A/RUNX2/OMD drives hRIFs to adopt osteogenic-like fates, by which OMD induces osteogenic-like microenvironment of renal interstitium to participate in RP formation. We identify OMD upregulation as a pathological feature of RP, paving the way for preventing CaOx stones., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Kidney stone disease (KSD) is a major public health concern associated with high morbidity and recurrence, places a significant burden on the health care system worldwide. Calcium oxalate (CaOx) alone or a mixture of CaOx and calcium phosphate stones accounting for more than 80 % of cases. However, beyond surgical removal, the prevention and reduction of recurrence of CaOx kidney stones have always been a challenge. Given that macrophages are traditional innate immune cells that play critical roles in the clearance of pathogens and the maintenance of tissue homeostasis, which have gained more and more interests in nephrolithiasis. Several studies recently clearly demonstrated that M2-macrophage could reduce the renal calcium oxalate (CaOx) crystal acumination, and provide premise insights and therapeutic options for KSD by modulating the macrophage phenotypes. However, the mechanism of macrophage-polarization regulation and that effects on kidney stone prevention and treatments are far from clear. Here, we comprehensively reviewed the literatures related to cytokines, epigenetic modifications and metabolic reprograming of macrophage in CaOx kidney stone disease, aimed to provide better understandings on macrophage polarization regulation as well as its potential clinical applications in CaOx kidney stone disease treatments and prevention., Competing Interests: Declaration of Competing Interest The authors declare that the results as reported in this manuscript are original and have not been published previously or accepted for publication elsewhere, and also not under consideration for publication elsewhere. All authors have seen and approved its publication. We also declare that there is no financial conflict of interest in this work., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Calcium oxalate (CaOx) kidney stones accumulate within the renal tubule due to high concentrations of insoluble deposits in the urine. Pb 2+ -induced Ca 2+ mobilization along with Pb 2+ -induced nephrotoxic effects within the proximal tubule have been well established; however, Pb 2+ mediated effects within the collecting duct remains insufficiently studied. Thus in vitro and ex vivo model systems were treated with increasing concentrations of lead (II) acetate (PbAc) ± sodium oxalate (Na 2 C 2 O 4 ) for 1 h, both individually and in combination. Pb 2+ -mediated solution turbidity increased 2 to 5 times greater post-exposure to 75, 100 and 200 μM Pb 2+ with the additional co-treatment of 10 mM oxalate in mouse inner medullary collecting duct (mIMCD-3) cells. Additionally, 100 μM and 200 μM Pb 2+ alone induced significant levels of intracellular Ca 2+ release. To validate Pb 2+ -mediated effects on the formation of CaOx crystals, alizarin red staining confirmed the presence of CaOx crystallization. Pb 2+ -induced intracellular Ca 2+ was also observed ex vivo in fly Malpighian tubules with significant increases in CaOx crystal formation via Pb 2+ -induced intracellular Ca 2+ release significantly increasing the average crystal number, size, and total area of crystal formation, which was ameliorated by tissue-specific SPoCk C transporter and Capa receptor knockdown. These studies demonstrate Pb 2+ -induced Ca 2+ release likely increases the formation of CaOx crystals, which is modulated by a G q -linked mechanism with concurrent Ca 2+ extracellular mobilization., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Greg M. Landry reports financial support was provided by American Association of Colleges of Pharmacy. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Calcium oxalate stone is the main type of kidney stone, so far there is no specific drug treatment. Here, we screened for oxalate-degrading probiotics and evaluated the potential preventive effect of lactic acid bacteria in rats with hyperoxaluria-induced kidney stones. The oxalate degradation efficiencies of the probiotics were determined to be 5-20% by in vitro experiments, of which the degradation efficiencies of Lactiplantibacillus plantarum AR342 and L. plantarum AR1089 were 17.32% and 14.15%, respectively. Through animal experiments, we found that L. plantarum AR1089 significantly attenuated kidney injury, as demonstrated by improving renal dysfunction and renal fibrosis, lowering creatinine and urea nitrogen levels. L. plantarum AR1089 was also effective in decreasing the number of calcium oxalate crystals in the urine and kidneys as well as ameliorating oxidative stress as evidenced by lowering the level of MDA and decreasing the level of SOD and CAT. Moreover, supplementation of L. plantarum AR1089 inhibited renal crystalline deposition by down-regulating the expression of KIM-1, OPN and MCP-1, and prevented hyperoxaluria-induced kidney stones by regulating the gut microbiota. Taken together, the present study shows that oral administration of L. plantarum AR1089, by attenuating kidney injury and regulating gut microbiota, is a potential therapy to reduce calcium oxalate crystals and prevent the progression of kidney stones.

Background: Kidney stone formation is a common disease that causes a significant threat to human health. The crystallization mechanism of calcium oxalate, the most common type of kidney stone, has been extensively researched, yet the damaging effects and mechanisms of calcium oxalate crystals on renal tubular epithelial cells remain incompletely elucidated. Regulated mitochondrial dynamics is essential for eukaryotic cells, but its role in the occurrence and progression of calcium oxalate (CaOx) nephrolithiasis is not yet understood., Methods: An animal model of calcium oxalate-related nephrolithiasis was established in adult male Sprague‒Dawley (SD) rats by continuously administering drinking water containing 1% ethylene glycol for 28 days. The impact of calcium oxalate crystals on mitochondrial dynamics and apoptosis in renal tubular epithelial cells was investigated using HK2 cells in vitro. Blood samples and bilateral kidney tissues were collected for histopathological evaluation and processed for tissue injury, inflammation, fibrosis, oxidative stress detection, and mitochondrial dynamics parameter analysis., Results: Calcium oxalate crystals caused higher levels of mitochondrial fission and apoptosis in renal tubular epithelial cells both in vivo and in vitro. Administration of a PPARγ agonist significantly alleviated mitochondrial fission and apoptosis in renal tubular epithelial cells, and improved renal function, accompanied by reduced levels of oxidative stress, increased antioxidant enzyme expression, alleviation of inflammation, and reduced fibrosis in vivo., Conclusion: Our results indicated that increased mitochondrial fission in renal tubular epithelial cells is a critical component of kidney injury caused by calcium oxalate stones, leading to the accumulation of reactive oxygen species within the tissue and the subsequent initiation of apoptosis. Regulating mitochondrial dynamics represents a promising approach for calcium oxalate nephrolithiasis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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 × 10 3 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 × 10 3 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., (© 2024. The Author(s).)

The interplay between crystals and epithelial cells forms the cornerstone of kidney stone development, communication between epithelial cells and macrophages emerging as a pivotal role in this process. We conducted next-generation sequencing on the secreted exosomes of TCMK-1 cells treated with calcium oxalate monohydrate (OX_EXO) or controls (NC_EXO), and on the macrophage cell line RAW264.7 stimulated with OX_EXO or NC_EXO, followed by validation of differentially expressed target proteins and miRNAs through Western blot and PCR. UPSET plots were employed to identify genes co-targeted by exosomal miRNAs. Various bioinformatic analyses were employed to predict potential mechanisms of the dysregulated genes. We integrated sequencing data from the GEO database, and validated findings using clinical patient urine and kidney tissues. We identified 665 differentially expressed exosomal miRNAs between OX_EXO and NC_EXO. Among the top 10 down-regulated miRNAs, the most targeted genes were AAK1 and NUFIP2, whereas PLCB1 was significantly targeted among the top 10 up-regulated miRNAs. In clinical specimens, we confirmed the differential expressions of five homologous miRNAs, as well as CNOT3, CNCNA1C, APEX1, and TMEM199. In conclusion, treatment of TCMK-1 cells with calcium oxalate significantly alerted the expression profile of exosomal miRNAs, subsequently influencing gene expression in macrophages, thereby modulating the processes of kidney stone formation., (© 2024. The Author(s).)

Calcium oxalate (CaOx) urolithiasis is a prevalent urinary disorder with significant clinical impact. This study investigates the therapeutic potential of Morin Hydrate (MH), a natural bioflavonoid, in preventing CaOx stone formation. Molecular docking studies revealed that MH binds strongly to glycolate oxidase (GO), suggesting its inhibitory effect on oxalate synthesis. In vitro assays demonstrated that MH effectively inhibits CaOx crystal nucleation, aggregation, and growth, altering crystal morphology to less stable forms. Diuretic activity studies in Wistar rats showed that MH substantially increased urine volume and ion excretion, indicating its moderate diuretic effect. In vivo experiments further supported these findings, with MH treatment improving urinary and serum markers, reducing oxidative stress, and protecting renal tissue, as evidenced by histopathological analysis. Notably, MH administration significantly decreased GO and lactate dehydrogenase activities in urolithiatic rats, indicating a reduction in oxalate production. These results suggest that MH is a promising candidate for the prevention and treatment of CaOx urolithiasis, with the potential for clinical application in reducing the risk and recurrence of kidney stones., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Calcium oxalate (CaOx) crystals are the main constituents of renal crystals in humans and induce tubular lumen damage in renal tubules, leading to renal calcium deposition and kidney stone formation. Oxidative stress and inflammation play important roles in regulating calcium oxalate-induced injury. Here, we evaluated the efficacy in inhibiting oxidation and inflammation of pectinolinarigenin, a biologically active natural metabolite, in CaOx nephrocalcinosis and further explored its targets of action. First, we developed cellular and mouse models of calcium oxalate renal nephrocalcinosis and identified the onset of oxidative stress and inflammation according to experimental data. We found that pectolinarigenin inhibited this onset while reducing renal crystal deposition. Network pharmacology was subsequently utilized to screen for hypoxia-inducible factor-1α (HIF-1α), a regulator involved in the body's release and over-oxidation of inflammatory factors. Finally, molecular docking, cellular thermal shift assay, and other experiments to detect HIF-1α expression showed that pectolinarigenin directly combined with HIF-1α and prevented downstream reactive oxygen species activation and release. Our results indicate that pectolinarigenin can target and inhibit HIF-1α-mediated inflammatory responses and oxidative stress damage and be a novel drug for CaOx nephrocalcinosis treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A retrospective study reported that empagliflozin reduced the risk of urinary stone events in patients with diabetes mellitus. To further investigate empagliflozin's potential, we conducted an animal experiment to determine whether empagliflozin can prevent renal stone formation in hyperoxaluria rats. Hyperoxaluria rat models were constructed by administrating 0.75 % ethylene glycol and 1 % ammonium chloride in water. The empagliflozin-treated rats were gauged with empagliflozin at different concentrations, and their body weight and blood sugar data were recorded. After 30 days of treatment, we obtained 24-h urine, kidney, and blood samples. The urine samples were subjected to component detection. Blood samples were prepared for component detection and cytokines detection. Renal samples were subjected to von Kossa staining, transmission electron microscopy, immunohistochemistry, and transcriptome sequencing analysis. Results showed that in empagliflozin-treated hyperoxaluria rats, renal crystal deposition and mitochondria injury, urinary concentration, and excretion of oxalate were significantly decreased. Additionally, plasma levels of VEGF, IL-2, IL-1β, and MCP-1 were decreased. Immunohistochemistry showed that renal expression of KIM-1, MCP-1 was significantly decreased in empagliflozin-treated hyperoxaluria rats. Transcriptome sequencing of renal tissue represented that 25 genes were down-regulated while 12 were up-regulated in empagliflozin-treated hyperoxaluria rats. These regulated genes were mainly enriched in fatty acid metabolism, insulin resistance, muscle contraction, bile secretion, and parathyroid metabolism. Our animal experiments found that empagliflozin could reduce urinary concentration and excretion of oxalate and inhibit renal inflammation, then abating renal calcium oxalate deposition in hyperoxaluria rats in a non-diabetic state., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objectives: To investigate the role of selenium and selenium-containing proteins in the etiology and pathogenesis of kidney stones. Methods: The HK-2 cell line was subjected to supersaturation oxalate treatment to establish an in vitro model of calcium oxalate kidney stones, while SD rats were administered with ethylene glycol to establish an in vivo model of calcium oxalate kidney stones. qPCR analysis was employed to investigate the alterations in selenoproteins within the models, and subsequently, genes exhibiting significant changes were identified. Subsequently, based on the functions of these genes, their regulatory effects on endoplasmic reticulum stress (ERS) and apoptosis during the disease progression were examined both in HK-2 cells and rat kidneys. Finally, Selenomethionine (SeMet) supplementation was introduced to explore its therapeutic potential for kidney stone management. Results: The involvement of Selenoprotein K in the pathogenesis of calcium oxalate kidney stone disease has been confirmed, exhibiting significant alterations. Manipulation of its expression levels through overexpression and knockdown techniques resulted in a corresponding reduction or increase in oxidative stress, ERS, and apoptosis within renal tubular epithelial cells. SelK regulates ERS and apoptosis by controlling the IRE1-ASK1-JNK pathway. In addition, SeMet treatment, which contains selenium, effectively reduced the levels of oxidative stress, ERS, and apoptosis in vivo and in vitro models, thereby alleviating tubular epithelial cell damage and reducing the formation of kidney stones in experimental rats. Discussion: Selenium is involved in the occurrence and development of kidney stones by regulating oxidative damage to renal tubular epithelial cells. The results suggest that dietary selenium supplementation in daily life may be of great significance for the prevention and treatment of kidney stones.

This study investigated the risk profile and the impact of dietary intervention in calcium oxalate stone formers with enteric hyperoxaluria under controlled, standardized conditions. Thirty-seven patients were included in the study. Dietary and 24-h urinary parameters were obtained on the self-selected diet and a balanced, standardized diet. Tests for [ 13 C 2 ]oxalate absorption, calcium- and ammonium chloride-loading were performed. Mean [ 13 C 2 ]oxalate absorption was 18.8%. A significant positive association was observed between urinary oxalate excretion and intestinal oxalate absorption. In addition, urinary oxalate excretion was significantly correlated with dietary oxalate intake. Mean urinary oxalate excretion decreased from 0.841 mmol/24 h on the usual diet to 0.662 mmol/24 h on the balanced diet, corresponding to a reduction of 21.3%. Besides hyperoxaluria, hypocitraturia and hypomagnesuria were the most common urinary abnormalities at baseline, being present in 83.8% and 81.1% of patients, respectively. Urinary citrate increased by 50.9% and magnesium excretion increased by 25.2% on the balanced diet. As a result, the relative supersaturation of calcium oxalate declined significantly (by 36.2%) on the balanced diet. Since 41% of patients on the balanced diet still had a urine volume of less than 2.0 L/24 h, efforts should be made to increase urine volume by increasing fluid intake and reducing intestinal fluid losses. Dietary intervention proved to be effective in reducing urinary oxalate excretion and should be a cornerstone of the treatment of patients with enteric hyperoxaluria.

Mitochondria are essential organelles because they generate the energy required for cellular functions. Kidney stones, as one of the most common urological diseases, have garnered significant attention. In this study, we first collected peripheral venous blood from patients with kidney stones and used qRT-PCR to detect mitochondrial DNA (mtDNA) copy number as a means of assessing mitochondrial function in these patients. Subsequently, through Western blotting, qPCR, immunofluorescence, immunohistochemistry, and transmission electron microscopy, we examined whether calcium oxalate crystals could cause mitochondrial dysfunction in the kidney in both in vitro and in vivo. We then examined the intersection of the DEGs obtained by transcriptome sequencing of the mouse kidney stone model with mitochondria-related genes, and performed KEGG and GO analyses on the intersecting genes. Finally, we administered the mitochondrial ROS scavenger Mito-Tempo in vivo and observed its effects. Our findings revealed that patients with kidney stones had a reduced mtDNA copy number in their peripheral venous blood compared to the control group, suggesting mitochondrial dysfunction in this population. This conclusion was further validated through in vitro and in vivo experiments. Enrichment analyses revealed that the intersecting genes were closely related to metabolism. We observed that after mitochondrial function was preserved, the deposition of calcium oxalate crystals decreased, and the kidney damage and inflammation caused by them were also alleviated. Our research indicates that kidney stones can cause mitochondrial dysfunction. After clearing mtROS, the damage and inflammation caused by kidney stones are reversed, providing new insights into the prevention and treatment of kidney stones., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The critical role of the human gut microbiota in kidney stone formation remains largely unknown, due to the low taxonomic resolution of previous sequencing technologies. Therefore, this study aimed to explore the gut microbiota using high-throughput sequencing to provide valuable insights and identify potential bacterial species and metabolite roles involved in kidney stone formation. The overall gut bacterial community and its potential functions in healthy participants and patients were examined using PacBio sequencing targeting the full-length 16S rRNA gene, coupled with stone and statistical analyses. Most kidney stones comprised calcium oxalate and calcium phosphate (75%), pure calcium oxalate (20%), and calcium phosphate and magnesium phosphate (5%), with higher content of Ca (130,510.5 ± 108,362.7 ppm) followed by P (18,746.4 ± 23,341.2 ppm). The microbial community structure was found to be weaker in patients' kidney stone samples, followed by patients' stool samples, than in healthy participants' stool samples. The most abundant bacterial species in kidney stone samples was uncultured Morganella, whereas that in patient and healthy participant stool samples was Bacteroides vulgatus. Similarly, Akkermansia muciniphila was significantly enriched in patient stool samples at the species level, whereas Bacteroides plebeius was significantly enriched in kidney stone samples than that in healthy participant stool samples. Three microbial metabolic pathways, TCA cycle, fatty acid oxidation, and urea cycle, were significantly enriched in kidney stone patients compared to healthy participants. Inferring bacteria at the species level revealed key players in kidney stone formation, enhancing the clinical relevance of gut microbiota., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Long non-coding ribose nucleic acids (lncRNAs) have been implicated in the development of nephrolithiasis. The study aims to investigate the interplay of lncRNA SBF2-AS1 (SETbinding factor 2 antisense RNA 1) and NLR family pyrin domain containing 3 (NLRP3) in regulating the calcium oxalate monohydrate (COM)-induced human kidney HK-2 cell injury. HK-2 cells were treated with COM (100 µg/mL) to create a cellular model of kidney injury. Gene and protein expression was assessed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blot. Proliferation and apoptosis rates, as well as levels of malondialdehyde (MDA), lactate dehydrogenase (LDH), superoxide dismutase (SOD), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 were measured. Additionally, potential miRNAs interacting with SBF2-AS1 and NLRP3 were predicted utilizing the starBase and TargetScan databases. The interference of SBF2-AS1 resulted in increased cell proliferation and SOD levels in HK-2 cells after COM induction. SBF2-AS1 silencing also reduced COM-induced cell death and inflammatory cytokine production by down-regulating NLRP3 protein expression. Conversely, forced upregulation of NLRP3 abrogated the effect of SBF2-AS1 interference. Notably, SBF2-AS1 interference on COM-induced oxidative stress and COM-induced cellular damage was rescued by antioxidant, indicating the involvement of oxidative burden in COM-induced damage. miR-302e acted as a mediator miRNA linking the functional association of SBF2-AS1 and NLRP3. Silencing SBF2-AS1 promoted miR-302e level and miR-302e reduced NLRP3 expression in HK-2 cells to protect against COM-induced damage. In summary, these findings suggest that downregulation of lncRNA SBF2-AS1 can potentially protect HK-2 cells from COM-induced injury by modulating the miR-302e/NLRP3 pathway., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Aims: Baicalin is a flavonoid derived from the root of the medicinal plant Scutellaria baicalensis Georgi (S. baicalensis) and is known for its various pharmacological properties. This study aimed to investigate the impact of baicalin (BAI) on the occurrence of kidney calcium oxalate crystal formation induced by ethylene glycol in male SD rats., Main Methods: A rat model of renal stones was created and various concentrations of baicalin were used for intervention. Samples of urine, blood, and kidney tissue were taken from the rats, and they were euthanized for biochemical and histopathological examinations., Key Findings: Our results show that baicalin treatment improved the weight loss induced by ethylene glycol (EG) and ammonium chloride (AC) in rats. Baicalin also reduced the formation of calcium oxalate crystals and protected kidney function in rats with urolithiasis. Furthermore, it lowered the level of malondialdehyde (MDA) and elevated the activity of antioxidant enzymes compared to the stone control group. Additionally, baicalin notably alleviated renal inflammation in rats with urolithiasis., Significance: The present study attributed clinical evidence first time that claiming the significant antiurolithic effect of baicalin and could be a cost-effective candidate for the prevention and treatment of urolithiasis., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Siqi Zhu reports financial support was provided by Guizhou Provincial Department of Science and Technology. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Ethnopharmacological Relevance: Calcium oxalate crystals play a key role in the development and recurrence of kidney stones (also known as urolithiasis); thus, inhibiting the formation of these crystals is a central focus of urolithiasis prevention and treatment. Previously, we reported the noteworthy in vitro inhibitory effects of Aspidopterys obcordata fructo oligosaccharide (AOFOS), an active polysaccharide of the traditional Dai medicine Aspidopterys obcordata Hemsl. (commonly known as Hei Gai Guan), on the growth of calcium oxalate crystals., Aim of the Study: To investigated the effectiveness and mechanism of AOFOS in treating kidney stones., Materials and Methods: A kidney stones rats model was developed, followed by examining AOFOS transport dynamics and effectiveness in live rats. Additionally, a correlation between the polysaccharide and calcium oxalate crystals was studied by combining crystallization experiments with density functional theory calculations., Results: The results showed that the polysaccharide was transported to the urinary system. Furthermore, their accumulation was inhibited by controlling their crystallization and modulating calcium ion and oxalate properties in the urine. Consequently, this approach helped effectively prevent kidney stone formation in the rats., Conclusions: The present study emphasized the role of the polysaccharide AOFOS in modulating crystal properties and controlling crystal growth, providing valuable insights into their potential therapeutic use in managing kidney stone formation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Accumulated evidences indicate that dysbiosis of the urinary microbiota is associated with kidney stone formation. In the present study, we aimed to investigate the urinary microbiota composition and functionality of patients with calcium oxalate stones and compare it with those of healthy individuals., Method: We collected bladder urine samples from 68 adult patients with calcium oxalate stones and 54 age-matched healthy controls by transurethral catheterization. 16S rRNA gene and shotgun sequencing were utilized to characterize the urinary microbiota and functionality associated with calcium oxalate stones., Results: After further exclusion, a total of 100 subjects was finally included and analyzed. The diversity of the urinary microbiota in calcium oxalate stone patients was not significantly different from that of healthy controls. However, the urinary microbiota structure of calcium oxalate stone formers significantly differed from that of healthy controls (PERMANOVA, r = 0.026, P = 0.019). Differential representation of bacteria (e.g., Bifidobacterium ) and several enriched functional pathways (e.g., threonine biosynthesis) were identified in the urine of calcium oxalate stone patients., Conclusion: Our results showed significantly different urinary microbiota structure and several enriched functional pathways in calcium oxalate stone patients, which provide new insight into the pathogenesis of calcium oxalate stones., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Xie, Zhang, Guo, Yuan, Xiao and Yuan.)

Kidney stones are a pervasive disease with notoriously high recurrence rates that require more effective treatment strategies. Herein, tartronic acid is introduced as an efficient inhibitor of calcium oxalate monohydrate (COM) crystallization, which is the most prevalent constituent of human kidney stones. A combination of in situ experimental techniques and simulations are employed to compare the inhibitory effects of tartronic acid with those of its molecular analogs. Tartronic acid exhibits an affinity for binding to rapidly growing apical surfaces of COM crystals, thus setting it apart from other inhibitors such as citric acid, the current preventative treatment for kidney stones. Bulk crystallization and in situ atomic force microscopy (AFM) measurements confirm the mechanism by which tartronic acid interacts with COM crystal surfaces and inhibits growth. These findings are consistent with in vivo studies that reveal the efficacy of tartronic acid is similar to that of citric acid in mouse models of hyperoxaluria regarding their inhibitory effect on stone formation and alleviating stone-related physical harm. In summary, these findings highlight the potential of tartronic acid as a promising alternative to citric acid for the management of calcium oxalate nephropathies, offering a new option for clinical intervention in cases of kidney stones., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Background: There are few reports of histopathology of any form of optic neuropathy. This article provides histopathologic findings of an adult-onset, nonprogressive optic neuropathy that was diagnosed clinically as nonacute, nonarteritic anterior ischemic optic neuropathy (NAION) but which was found by a pathological study to be associated with diffuse calcium oxalosis that was confined in the involved orbit., Methods: This is a case report that includes results of a neuro-ophthalmologic examination and histopathology of a complete autopsy, including en bloc removal of both orbits and the brain. The unaffected orbit/optic nerve served as a control. The affected orbit was serially sectioned into 2,550 increments each separated by 10 μm; the uninvolved orbit was sectioned into 150 equally spaced sections. The main outcome measures were derived from the autopsy, especially from the thin-section histopathologic study of both orbits that focused on blood vessels and the site of neural damage within the optic nerve., Results: The neuro-ophthalmologic examination revealed a unilateral optic neuropathy with pallor of the left optic nerve head that had been documented just before death. The general autopsy showed acute bacterial endocarditis and a recent cerebral hematoma that caused death. Histopathology revealed sectoral loss of optic nerve axons in the left eye. Numerous arterial walls in the left orbit, including short posterior ciliary arteries and the central retinal artery, contained hundreds of crystals with anisotropic, colorful birefringence consistent with calcium oxalosis. Crystals were not found in the right, control orbit or elsewhere in the body., Conclusions: The patient developed an optic neuropathy late in life that was diagnosed by an experienced neuro-ophthalmologist as being most consistent with nonacute, nonarteritic anterior ischemic optic neuropathy. The autopsy identified sectoral loss of optic nerve fibers consistent with that diagnosis. However, the unexpected discovery of calcium oxalate crystals in blood vessels of the involved orbit, which curiously were not present elsewhere in the body, raises a question of their etiological role in this particular optic neuropathy. Whether the crystals were causal, epiphenomenal, or purely incidental to the optic neuropathy cannot be answered by our study., Competing Interests: The authors report no conflicts of interest., (Copyright © 2023 by North American Neuro-Ophthalmology Society.)

The inhibition of cell surface crystal adhesion and an appropriate increase in crystal endocytosis contribute to the inhibition of kidney stone formation. In this study, we investigated the effects of different degrees of carboxymethylation on these processes. An injury model was established by treating human renal proximal tubular epithelial (HK-2) cells with 98.3 ± 8.1 nm calcium oxalate dihydrate (nanoCOD) crystals. The HK-2 cells were protected with carboxy (-COOH) Desmodium styracifolium polysaccharides at 1.17% (DSP0), 7.45% (CDSP1), 12.2% (CDSP2), and 17.7% (CDSP3). Changes in biochemical indexes and effects on nanoCOD adhesion and endocytosis were detected. The protection of HK-2 cells from nanoCOD-induced oxidative damage by carboxymethylated Desmodium styracifolium polysaccharides (CDSPs) is closely related to the protection of subcellular organelles, such as mitochondria. CDSPs can reduce crystal adhesion on the cell surface and maintain appropriate crystal endocytosis, thereby reducing the risk of kidney stone formation. CDSP2 with moderate -COOH content showed the strongest protective activity among the CDSPs., (© 2024 Wiley Periodicals LLC.)

Urolithiasis is a high‑incidence disease caused by calcium oxalate (mainly), uric acid, calcium phosphate, struvite, apatite, cystine and other stones. The development of kidney stones is closely related to renal tubule cell damage and crystal adhesion and aggregation. Cell death, comprising the core steps of cell damage, can be classified into various types (i.e., apoptosis, ferroptosis, necroptosis and pyroptosis). Different crystal types, concentrations, morphologies and sizes cause tubular cell damage via the regulation of different forms of cell death. Oxidative stress caused by high oxalate or crystal concentrations is considered to be a precursor to a variety of types of cell death. In addition, complex crosstalk exists among numerous signaling pathways and their key molecules in various types of cell death. Urolithiasis is considered a metabolic disorder, and tricarboxylic acid cycle‑related molecules, such as citrate and succinate, are closely related to cell death and the inhibition of stone development. However, a literature review of the associations between kidney stone development, metabolism and various types of cell death is currently lacking, at least to the best of our knowledge. Thus, the present review summarizes the major advances in the understanding of regulated cell death and urolithiasis progression.

The engineered clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system is currently widely applied in genetic editing and transcriptional regulation. The catalytically inactivated CasRx (dCasRx) has the ability to selectively focus on the mRNA coding region without disrupting transcription and translation, opening up new avenues for research on RNA modification and protein translation control. This research utilized dCasRx to create a translation-enhancement system for mammals called dCasRx-eIF4GI, which combined eukaryotic translation initiation factor 4G (eIF4GI) to boost translation levels of the target gene by recruiting ribosomes, without affecting mRNA levels, ultimately increasing translation levels of different endogenous proteins. Due to the small size of dCasRx, the dCasRx-eIF4GI translation enhancement system was integrated into a single viral vector, thus optimizing the delivery and transfection efficiency in subsequent applications. Previous studies reported that ferroptosis, mediated by calcium oxalate (CaOx) crystals, significantly promotes stone formation. In order to further validate its developmental potential, it was applied to a kidney stone model in vitro and in vivo. The manipulation of the ferroptosis regulatory gene FTH1 through single-guide RNA (sgRNA) resulted in a notable increase in FTH1 protein levels without affecting its mRNA levels. This ultimately prevented intracellular ferroptosis and protected against cell damage and renal impairment caused by CaOx crystals. Taken together, this study preliminarily validated the effectiveness and application prospects of the dCasRx-eIF4GI translation enhancement system in mammalian cell-based disease models, providing novel insights and a universal tool platform for protein translation research and future therapeutic approaches for nephrolithiasis., (© 2024. The Author(s).)

C-C chemokine receptor type 2 (CCR2) is a monocyte chemokine associated with oxidative stress and inflammation. Kidney stones (KS) are composed of calcium oxalate (CaOx), which trigger renal oxidative stress and inflammatory. This study aims to evaluate the effects of CCR2 on KS in vivo and in vitro. Eight-week-old male C57BL/6J mice were intraperitoneally injected with glyoxylate (GOX) daily to establish a KS model, and along with CCR2 antagonist (INCB3344) treatment on days 2, 4, and 6. The results showed that CCR2 antagonist reduced renal injury markers (blood urea nitrogen and serum creatinine), alleviated renal tubular injury and CaOx crystal deposition. CCR2 antagonist also decreased CCR2 expression induced by GOX treatment and increased Nrf2 expression. GOX treatment promoted malondialdehyde (MDA) production, decreased glutathione (GSH) content, and inhibited catalase (CAT) and superoxide dismutase (SOD) activity, however, CCR2 antagonist attenuated the above effects of GOX. CCR2 antagonist had inhibitory effects on GOX-induced inflammatory cytokine expression (IL1B, IL6 and MCP1), and inhibited apoptosis by increasing Bcl-2 expression and decreasing Bax and cleaved-caspase 3 expression. In vitro experiments were performed by co-culture model of CaOx-induced damaged HK-2 cells and macrophage-like THP-1 cells. CCR2 antagonist inhibited CaOx-induced THP-1 cell M1 polarization by decreasing the TNF-α, IL6 and iNOS levels, and further alleviated CaOx-induced oxidative stress damage, inflammatory response and apoptosis of HK-2 cells. The study suggests that CCR2 antagonist may be resistant to CaOx crystals-induced oxidative stress and inflammation by inhibiting macrophage M1 polarization.

The CRISPR-Cas system, initially for DNA-level gene editing and transcription regulation, has expanded to RNA targeting with the Cas13d family, notably the RfxCas13d. This advancement allows for mRNA targeting with high specificity, particularly after catalytic inactivation, broadening the exploration of translation regulation. This study introduces a CRISPR-dCas13d-eIF4G fusion module, combining dCas13d with the eIF4G translation regulatory element, enhancing target mRNA translation levels. This module, using specially designed sgRNAs, selectively boosts protein translation in targeted tissue cells without altering transcription, leading to notable protein expression upregulation. This system is applied to a kidney stone disease model, focusing on ferroptosis-linked GPX4 gene regulation. By targeting GPX4 with sgRNAs, its protein expression is upregulated in human renal cells and mouse kidney tissue, countering ferroptosis and resisting calcium oxalate-induced cell damage, hence mitigating stone formation. This study evidences the CRISPR-dCas13d-eIF4G system's efficacy in eukaryotic cells, presenting a novel protein translation research approach and potential kidney stone disease treatment advancements., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Sex differences in kidney stone formation are well known. Females generally have slightly acidic blood and higher urine pH when compared with males, which makes them more vulnerable to calcium stone formation, yet the mechanism is still unclear. We aimed to examine the role of sex in stone formation during hypercalciuria and urine alkalinization through acetazolamide and calcium gluconate supplementation, respectively, for 4 weeks in wild-type (WT) and moderately hypercalciuric [TRPC3 knockout [KO](-/-)] male and female mice. Our goal was to develop calcium phosphate (CaP) and CaP+ calcium oxalate mixed stones in our animal model to understand the underlying sex-based mechanism of calcium nephrolithiasis. Our results from the analyses of mice urine, serum, and kidney tissues show that female mice (WT and KO) produce more urinary CaP crystals, higher [Ca 2+ ], and pH in urine compared to their male counterparts. We identified a sex-based relationship of stone-forming phenotypes (types of stones) in our mice model following urine alkalization/calcium supplementation, and our findings suggest that female mice are more susceptible to CaP stones under those conditions. Calcification and fibrotic and inflammatory markers were elevated in treated female mice compared with their male counterparts, and more so in TRPC3 KO mice compared with their WT counterparts. Together these findings contribute to a mechanistic understanding of sex-influenced CaP and mixed stone formation that can be used as a basis for determining the factors in sex-related clinical studies., (Published by Elsevier Inc.)

Formation of calcium oxalate (CaOx) crystal, the most common composition in kidney stones, occurs following supersaturation of calcium and oxalate ions in the urine. In addition to endogenous source, another main source of calcium and oxalate ions is dietary intake. In the intestinal lumen, calcium can bind with oxalate to form precipitates to be eliminated with feces. High intake of oxalate-rich foods, inappropriate amount of daily calcium intake, defective intestinal transporters for oxalate secretion and absorption, and gastrointestinal (GI) malabsorption (i.e., from gastric bypass surgery) can enhance intestinal oxalate absorption, thereby increasing urinary oxalate level and risk of kidney stone disease (KSD). The GI microbiome rich with oxalate-degrading bacteria can reduce intestinal oxalate absorption and urinary oxalate level. In addition to the oxalate-degrading ability, the GI microbiome also affects expression of oxalate transporters and net intestinal oxalate transport, cholesterol level, and short-chain fatty acids (SCFAs) production, leading to lower KSD risk. Recent evidence also shows beneficial effects of urinary microbiome in KSD prevention. This review summarizes the current knowledge on the aforementioned aspects. Potential benefits of the GI and urinary microbiomes as probiotics for KSD prevention are emphasized. Finally, challenges and future perspectives of probiotic treatment in KSD are discussed., Competing Interests: Conflicts of Interest The authors declare NO conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Enteric hyperoxaluria is a metabolic disorder resulting from conditions associated with fatty acid malabsorption and characterized by an increased urinary output of oxalate. Oxalate is excessively absorbed in the gut and then excreted in urine where it forms calcium oxalate crystals, inducing kidney stones formation and crystalline nephropathies. Enteric hyperoxaluria is probably underdiagnosed and may silently damage kidney function of patients affected by bowel diseases. Moreover, the prevalence of enteric hyperoxaluria has increased because of the development of bariatric surgical procedures. Therapeutic options are based on the treatment of the underlying disease, limitation of oxalate intakes, increase in calcium salts intakes but also increase in urine volume and correction of hypocitraturia. There are few data regarding the natural evolution of kidney stone events and chronic kidney disease in these patients, and there is a need for new treatments limiting kidney injury by calcium oxalate crystallization., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: EL and MD filed a patent relative to the use of Stiripentol against primary hyperoxaluria (WO2017140658A1). The authors declare no competing interest regarding this review., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

In calcium nephrolithiasis (CaNL), most calcium kidney stones are identified as calcium oxalate (CaOx) with variable amounts of calcium phosphate (CaP), where CaP is found as the core component. The nucleation of CaP could be the first step of CaP+CaOx (mixed) stone formation. High urinary supersaturation of CaP due to hypercalciuria and an elevated urine pH have been described as the two main factors in the nucleation of CaP crystals. Our previous in vivo findings (in mice) show that transient receptor potential canonical type 3 (TRPC3)-mediated Ca 2+ entry triggers a transepithelial Ca 2+ flux to regulate proximal tubular (PT) luminal [Ca 2+ ], and TRPC3-knockout (KO; -/-) mice exhibited moderate hypercalciuria and microcrystal formation at the loop of Henle (LOH). Therefore, we utilized TRPC3 KO mice and exposed them to both hypercalciuric [2% calcium gluconate (CaG) treatment] and alkalineuric conditions [0.08% acetazolamide (ACZ) treatment] to generate a CaNL phenotype. Our results revealed a significant CaP and mixed crystal formation in those treated KO mice (KOT) compared to their WT counterparts (WTT). Importantly, prolonged exposure to CaG and ACZ resulted in a further increase in crystal size for both treated groups (WTT and KOT), but the KOT mice crystal sizes were markedly larger. Moreover, kidney tissue sections of the KOT mice displayed a greater CaP and mixed microcrystal formation than the kidney sections of the WTT group, specifically in the outer and inner medullary and calyceal region; thus, a higher degree of calcifications and mixed calcium lithiasis in the kidneys of the KOT group was displayed. In our effort to find the Ca 2+ signaling pathophysiology of PT cells, we found that PT cells from both treated groups (WTT and KOT) elicited a larger Ca 2+ entry compared to the WT counterparts because of significant inhibition by the store-operated Ca 2+ entry (SOCE) inhibitor, Pyr6. In the presence of both SOCE (Pyr6) and ROCE (receptor-operated Ca 2+ entry) inhibitors (Pyr10), Ca 2+ entry by WTT cells was moderately inhibited, suggesting that the Ca 2+ and pH levels exerted sensitivity changes in response to ROCE and SOCE. An assessment of the gene expression profiles in the PT cells of WTT and KOT mice revealed a safeguarding effect of TRPC3 against detrimental processes (calcification, fibrosis, inflammation, and apoptosis) in the presence of higher pH and hypercalciuric conditions in mice. Together, these findings show that compromise in both the ROCE and SOCE mechanisms in the absence of TRPC3 under hypercalciuric plus higher tubular pH conditions results in higher CaP and mixed crystal formation and that TRPC3 is protective against those adverse effects.

Primary hyperoxalurias (PHs) are inherited metabolic disorders marked by enzymatic cascade disruption, leading to excessive oxalate production that is subsequently excreted in the urine. Calcium oxalate deposition in the renal tubules and interstitium triggers renal injury, precipitating systemic oxalate build-up and subsequent secondary organ impairment. Recent explorations of novel therapeutic strategies have challenged and necessitated the reassessment of established management frameworks. The execution of diverse clinical trials across various medication classes has provided new insights and knowledge. With the evolution of PH treatments reaching a new milestone, prompt and accurate diagnosis is increasingly critical. Developing early, effective management and treatment plans is essential to improve the long-term quality of life for PH patients.

Several mechanisms underlying nephrolithiasis, one of the most common urological diseases, involve calcium oxalate formation, including oxidative stress, inflammatory reactions, fibrosis, pyroptosis, and apoptosis. Although lycopene has strong antioxidant activity, its protective effects against CaOx-induced injury have not yet been reported. This study aimed to systematically investigate the protective effects of lycopene and explore its mechanisms and molecular targets. Crystal deposition, renal function, oxidative stress, inflammatory response, fibrosis, pyroptosis, and apoptosis were assessed to evaluate the renoprotective effects of lycopene against crystal formation in a CaOx rat model and oxalate-stimulated NRK-52E and HK-2 cells. Lycopene markedly ameliorated crystal deposition, restored renal function, and suppressed kidney injury by reducing oxidative stress, apoptosis, inflammation, fibrosis, and pyroptosis in the rats. In cell models, lycopene pretreatment reversed reactive oxygen species increase, apoptotic damage, intracellular lactate dehydrogenase release, cytotoxicity, pyroptosis, and extracellular matrix deposition. Network pharmacology and proteomic analyses were performed to identify lycopene target proteins under CaOx-exposed conditions, and the results showed that Trappc4 might be a pivotal target gene for lycopene, as identified by cellular thermal shift assay and surface plasmon resonance analyses. Based on molecular docking, molecular dynamics simulations, alanine scanning mutagenesis, and saturation mutagenesis, we observed that lycopene directly interacts with Trappc4 via hydrophobic bonds, which may be attributed to the PHE4 and PHE142 residues, preventing ERK1/2 or elevating AMPK signaling pathway phosphorylation events. In conclusion, lycopene might ameliorate oxalate-induced renal tubular epithelial cell injury via the Trappc4/ERK1/2/AMPK pathway, indicating its potential for the treatment of nephrolithiasis.

Needle-like calcium oxalate crystals called raphides are unique structures in the plant kingdom. Multiple biomacromolecules work together in the regulatory and transportation pathways to form raphides; however, the mechanism by which this occurs remains unknown. Using banana (Musa spp.), this study combined in vivo methods including confocal microscopy, transmission electron microscopy, and Q Exactive mass spectrometry to identify the main biomolecules, such as vesicles, together with the compositions of lipids and proteins in the crystal chamber, which is the membrane compartment that surrounds each raphide during its formation. Simulations of the vesicle transportation process and the synthesis of elongated calcium oxalate crystals in vitro were then conducted, and the results suggested that the vesicles carrying amorphous calcium oxalate and proteins embedded in raphides are transported along actin filaments. These vesicles subsequently fuse with the crystal chamber, utilizing the proteins embedded in the raphides as a template for the final formation of the structure. Our findings contribute to the fundamental understanding of the regulation of the diverse biomacromolecules that are crucial for raphide formation. Moreover, the implications of these findings extend to other fields such as materials science, and particularly the synthesis of functionalized materials., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Calcium oxalate monohydrate (COM) forms the most common type of kidney stones observed in clinics, elevated levels of urinary oxalate being the principal risk factor for such an etiology. The objective of the present study was to evaluate the anti-nephrolithiatic effect of herbo-mineral formulation, Lithom., Methods: The in vitro biochemical synthesis of COM crystals in the presence of Lithom was performed and observations were made by microscopy and Scanning Electron Microscope (SEM) based analysis for the detection of crystal size and morphology. The phytochemical composition of Lithom was evaluated by Ultra-High-Performance Liquid Chromatography (UHPLC). The in vivo model of Ethylene glycol-induced hyperoxaluria in Sprague-Dawley rats was used for the evaluation of Lithom. The animals were randomly allocated to 5 different groups namely Normal control, Disease control (ethylene glycol (EG), 0.75%, 28 days), Allopurinol (50 mg/kg, q.d. ), Lithom (43 mg/kg, b.i.d. ), and Lithom (129 mg/kg, b.i.d. ). Analysis of crystalluria, oxalate, and citrate levels, oxidative stress parameters (malondialdehyde (MDA), catalase, myeloperoxidase (MPO)), and histopathology by hematoxylin and eosin (H&E) and Von Kossa staining was performed for evaluation of Lithom., Results: The presence of Lithom during COM crystals synthesis significantly reduced the average crystal area, feret's diameter, and area-perimeter ratio, in a dose-dependent manner. SEM analysis revealed that COM crystals synthesized in the presence of 100 and 300 μg/mL of Lithom exhibited a veritable morphological transition from irregular polygons with sharp edges to smoothened smaller cuboid polygons. UHPLC analysis of Lithom revealed the presence of Trigonelline, Bergenin, Xanthosine, Adenosine, Bohoervinone B, Vanillic acid, and Ellagic acid as key phytoconstituents. In EG-induced SD rats, the Lithom-treated group showed a decrease in elevated urinary oxalate levels, oxidative stress, and renal inflammation. Von Kossa staining of kidney tissue also exhibited a marked reduction in crystal depositions in Lithom-treated groups., Conclusion: Taken together, Lithom could be a potential clinical-therapeutic alternative for management of nephrolithiasis.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Macrophages play a role in nephrolithiasis, offering the possibility of developing macrophage-mediated preventive therapies. To establish a system for screening drugs that could prevent the formation of kidney stones, we aimed to develop a model using human induced pluripotent stem cell (iPSC)-derived macrophages to study phagocytosis of calcium oxalate monohydrate (COM) crystals. Human iPSCs (201B7) were cultured. CD14+ monocytes were recovered using a stepwise process that involved the use of growth factors and cytokines. These cells were then allowed to differentiate into M1 and M2 macrophages. The macrophages were co-cultured with COM crystals and used in the phagocytosis experiments. Live cell imaging and polarized light observation via super-resolution microscopy were used to visualize phagocytosis. Localization of phagocytosed COM crystals was observed using transmission electron microscopy. Intracellular fluorescence intensity was measured using imaging cytometry to quantify phagocytosis. Human iPSCs successfully differentiated into M1 and M2 macrophages. M1 macrophages adhered to the culture plate and moved COM crystals from the periphery to cell center over time, whereas M2 macrophages did not adhere to the culture plate and actively phagocytosed the surrounding COM crystals. Fluorescence assessment over a 24-h period showed that M2 macrophages exhibited higher intracellular fluorescence intensity (5.65-times higher than that of M1 macrophages at 4.5 h) and maintained this advantage for 18 h. This study revealed that human iPSC-derived macrophages have the ability to phagocytose COM crystals, presenting a new approach for studying urinary stone formation and highlighting the potential of iPSC-derived macrophages as a tool to screen nephrolithiasis-related drugs., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Oxidative damage to the kidneys is a primary factor in the occurrence of kidney stones. This study explores the inhibitory effect of Porphyra yezoensis polysaccharides (PYP) on oxalate-induced renal injury by detecting levels of oxidative damage, expression of adhesion molecules, and damage to intracellular organelles and revealed the molecular mechanism by molecular biology methods. Additionally, we validated the role of PYP in vivo using a crystallization model of hyperoxalate-induced rats. PYP effectively scavenged the overproduction of reactive oxygen species (ROS) in HK-2 cells, inhibited the adhesion of calcium oxalate (CaOx) crystals on the cell surface, unblocked the cell cycle, restored the depolarization of the mitochondrial membrane potential, and inhibited cell death. PYP upregulated the expression of antioxidant proteins, including Nrf2, HO-1, SOD, and CAT, while decreasing the expression of Keap-1, thereby activating the Keap1/Nrf2 signaling pathway. PYP inhibited CaOx deposition in renal tubules in the rat crystallization model, significantly reduced high oxalate-induced renal injury, decreased the levels of the cell surface adhesion proteins, improved renal function in rats, and ultimately inhibited the formation of kidney stones. Therefore, PYP, which has crystallization inhibition and antioxidant properties, may be a therapeutic option for the treatment of kidney stones.

Ferroptosis is a specific type of programmed cell death characterized by iron-dependent lipid peroxidation. Understanding the involvement of ferroptosis in calcium oxalate (CaOx) stone formation may reveal potential targets for this condition. The publicly available dataset GSE73680 was used to identify 61 differentially expressed ferroptosis-related genes (DEFERGs) between normal kidney tissues and Randall's plaques (RPs) from patients with nephrolithiasis through employing weighted gene co-expression network analysis (WGCNA). The findings were validated through in vitro and in vivo experiments using CaOx nephrolithiasis rat models induced by 1% ethylene glycol administration and HK-2 cell models treated with 1 mM oxalate. Through WGCNA and the machine learning algorithm, we identified LAMP2 and MDM4 as the hub DEFERGs. Subsequently, nephrolithiasis samples were classified into cluster 1 and cluster 2 based on the expression of the hub DEFERGs. Validation experiments demonstrated decreased expression of LAMP2 and MDM4 in CaOx nephrolithiasis animal models and cells. Treatment with ferrostatin-1 (Fer-1), a ferroptosis inhibitor, partially reversed oxidative stress and lipid peroxidation in CaOx nephrolithiasis models. Moreover, Fer-1 also reversed the expression changes of LAMP2 and MDM4 in CaOx nephrolithiasis models. Our findings suggest that ferroptosis may be involved in the formation of CaOx kidney stones through the regulation of LAMP2 and MDM4.

This study was aimed to investigate the preventive effects of N-acetyl-L-cysteine (NAC) against renal tubular cell injury induced by oxalate and stone formation and further explore the related mechanism. Transcriptome sequencing combined with bioinformatics analysis were performed to identify differentially expressed gene (DEG) and related pathways. HK-2 cells were pretreated with or without antioxidant NAC/with or silencing DEG before exposed to sodium oxalate. Then, the cell viability, oxidative biomarkers of superoxidase dismutase (SOD) and malondialdehyde (MDA), apoptosis and cell cycle were measured through CCK8, ELISA and flow cytometry assay, respectively. Male SD rats were separated into control group, hyperoxaluria (HOx) group, NAC intervention group, and TGF-β/SMAD pathway inhibitor group. After treatment, the structure changes and oxidative stress and CaOx crystals deposition were evaluated in renal tissues by H&E staining, immunohistochemical and Pizzolato method. The expression of TGF-β/SMAD pathway related proteins (TGF-β1, SMAD3 and SMAD7) were determined by Western blot in vivo and in vitro. CDKN2B is a DEG screened by transcriptome sequencing combined with bioinformatics analysis, and verified by qRT-PCR. Sodium oxalate induced declined HK-2 cell viability, in parallel with inhibited cellular oxidative stress and apoptosis. The changes induced by oxalate in HK-2 cells were significantly reversed by NAC treatment or the silencing of CDKN2B. The cell structure damage and CaOx crystals deposition were observed in kidney tissues of HOx group. Meanwhile, the expression levels of SOD and 8-OHdG were detected in kidney tissues of HOx group. The changes induced by oxalate in kidney tissues were significantly reversed by NAC treatment. Besides, expression of SMAD7 was significantly down-regulated, while TGF-β1 and SMAD3 were accumulated induced by oxalate in vitro and in vivo. The expression levels of TGF-β/SMAD pathway related proteins induced by oxalate were reversed by NAC. In conclusion, we found that NAC could play an anti-calculus role by mediating CDKN2B/TGF-β/SMAD axis., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The study subject was a healthy, 47-year-old man, a low temperature Guinness World Record holder. He spent 50 days alone in Rovaniemi, Lapland, and functioned in the ambient temperature ranging from +2°C to -37°C. He did not use sources of heat, he did not eat warm meals or drink hot water, and did not dry his clothes. He slept in an igloo, on an ice cover of 20-30 cm. He spent 10 hours a day in a sleeping bag and for the remaining time he walked, skied, or rode a bicycle, and practiced swimming. The aim of the study was a laboratory assessment of renal capacity in a man exposed to long-term extremely low ambient temperatures. The study was approved by the Ethical Committee at the Regional Medical Chamber in Krakow, Poland (approval No.: 194/KBL/OIL/2019). Twice during the observation, urine and blood were collected and analyzed: before and after the prolonged exposure to extremely low ambient temperatures. Changes were seen in many blood and urine parameters, but in urine, they were more significant. In urine, decreased values of sodium (by 53.9%), potassium (by 22.6%), creatinine (by 65.5%), urea (by 61.3%), uric acid (by 58.4%), and protein (by 50%) were observed. Neutrophil gelatinase-associated lipocalin (NGAL) increased by 34%. Absence of calcium oxalate excretion was reported relative to the value before the exposure to cold. In blood, increased values of interleukin-6 (by 60%) and β-2-microglobulin (by 26.9%) were observed. Erythropoietin decreased by 22.4%. No changes were noted in estimated glomerular filtration rate. The study subject lost 10 kg in weight. On the basis of the results obtained during the observation, it can be determined that the probable cause of changes in the laboratory results of the subject was the diet used, and not a dysfunction of the excretory system. The body weight loss and activation of compensating mechanisms focused on saving vitally important diet components, caused by the insufficient diet, exclude the theory of a negative effect of exposure to extremely low temperatures on renal filtration function.

The study investigated calcium oxalate (CaOx) crystal composition, accumulation, synthesis, and degradation in five rhubarb species from the North-Western Indian Himalayas. Techniques like optical and scanning electron microscopy (SEM), SEM-energy-dispersive X-ray spectroscopy (SEM-EDS), inductively coupled plasma-optical emission spectrometry (ICP-OES), X-ray diffraction spectroscopy (XRD), and real-time (qRT-PCR) expression analysis of strategic genes were used to understand the processes of oxalate synthesis and precipitation. Results showed crystals tend to accumulate around vascular bundles in all species, irrespective of size, indicating a consistent pattern. Crystal synthesis and accumulation were stress-driven, linked to substrate composition, and in planta soluble oxalate and calcium levels, paralleling oxalate precursors. Based on their availability, CaOx crystals precipitated heavy metals mostly associated with its weddellite form. Crystal content correlated positively with mRNA levels of calcium/oxalate/ascorbate-related and stress-responsive genes, and negatively with oxalate oxidation/decarboxylation genes. CaOx crystals were suggested as potential biominerals for addressing heavy metal stress in agriculture., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)