The identification of key molecules and pathways in the crosstalk of calcium oxalate-treated TCMK-1 cells and macrophage via exosomes.

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.

Research progress on miR-124-3p in the field of kidney disease.

MicroRNAs (miRNAs) are 18-25 nucleotides long, single-stranded, non-coding RNA molecules that regulate gene expression. They play a crucial role in maintaining normal cellular functions and homeostasis in organisms. Studies have shown that miR-124-3p is highly expressed in brain tissue and plays a significant role in nervous system development. It is also described as a tumor suppressor, regulating biological processes like cancer cell proliferation, apoptosis, migration, and invasion by controlling multiple downstream target genes. miR-124-3p has been found to be involved in the progression of various kidney diseases, including diabetic kidney disease, calcium oxalate kidney stones, acute kidney injury, lupus nephritis, and renal interstitial fibrosis. It mediates these processes through mechanisms like oxidative stress, inflammation, autophagy, and ferroptosis. To lay the foundation for future therapeutic strategies, this research group reviewed recent studies on the functional roles of miR-124-3p in renal diseases and the regulation of its downstream target genes. Additionally, the feasibility, limitations, and potential application of miR-124-3p as a diagnostic biomarker and therapeutic target were thoroughly investigated.

A high-calcium environment induced ectopic calcification of renal interstitial fibroblasts via TFPI-2-DCHS1-ALP/ENPP1 axis to participate in Randall's plaque formation.

Randall's plaques (RP) serve as anchoring sites for calcium oxalate (CaOx) stones, but the underlying mechanism remains unclear. Renal interstitium with a high-calcium environment is identified as pathogenesis of RP formation where the role of human renal interstitial fibroblasts (hRIFs) was highlighted. Our study aims to elucidate the potential mechanism by which a high-calcium environment drives ectopic calcification of hRIFs to participate in RP formation. Alizarin Red staining demonstrated calcium nodules in hRIFs treated with high-calcium medium. Utilizing transcriptome sequencing, tissue factor pathway inhibitor-2 (TFPI-2) was found to be upregulated in high-calcium-induced hRIFs and RP tissues, and TFPI-2 promoted high-calcium-induced calcification of hRIFs. Subsequently, the downstream regulator of TFPI2 was screened by transcriptome sequencing analysis of hRIFs with TFPI-2 knockdown or overexpressed. Dachsous Cadherin Related 1 (DCHS1) knockdown was identified to suppress the calcification of hRIFs enhanced by TFPI-2. Further investigation revealed that TFPI-2/DCHS1 axis promoted high-calcium-induced calcification of hRIFs via disturbing the balance of ENPP1/ALP activities, but without effect on the canonical osteogenic markers, such as osteopontin (OPN), osteogenic factors runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2). In summary, our study mimicked the high-calcium environment observed in CaOx stone patients with hypercalciuria, and discovered that the high-calcium drove ectopic calcification of hRIFs via a novel TFPI-2-DCHS1-ALP/ENPP1 pathway rather than adaption of osteogenic phenotypes to participate in RP formation.

Association between oxidative balance score and kidney stones: data from the national health and nutrition examination survey (NHANES).

PURPOSE: Some studies have found that the pathological formation of kidney stones is closely related to injury and inflammatory response. Behaviors such as dietary composition, physical activity, obesity and smoking can all affect the body's oxidative stress levels. In order to evaluate the effects of various diets and lifestyles on the body's oxidative and antioxidant systems, an oxidative balance score was developed. To investigate whether the OBS is associated with the development of kidney stones. METHODS: Data were taken from the National Health and Nutrition Examination Survey (NHANES) from 2007-2018, followed by retrospective observational studies. The association between kidney stones and OBS was analyzed using survey-weighted logistic regression by adjusting for demographics, laboratory tests, and medical comorbidity covariates. The oxidative balance score is calculated by screening 16 nutrients and 4 lifestyle factors, including 5 prooxidants and 15 antioxidants, based on prior information about the relationship between oxidation levels in the body and nutrients or lifestyle factors. RESULTS: A total of 26,786 adult participants were included in the study, of which 2,578, or 9.62%, had a history of nephrolithiasis. Weighted logistic regression analysis found an association between OBS and kidney stones. In the fully tuned model, i.e., model 3, the highest quartile array of OBS was associated with the lowest quartile array of OBS (OR = 0.73 (0.57, 0.92)) with the risk of kidney stone (p = 0.01), and was statistically significant and remained relatively stable in each model. At the same time, the trend test in the model is also statistically significant. With the increase of OBS, the OR value of kidney stones generally tends to decrease. CONCLUSIONS: There is an inverse correlation between OBS and kidney stone disease. At the same time, higher OBS suggests that antioxidant exposure is greater than pro-oxidative exposure in diet and lifestyle, and is associated with a lower risk of kidney stones.

Understanding formation processes of calcareous nephrolithiasis in renal interstitium and tubule lumen.

Kidney stone, one of the oldest known diseases, has plagued humans for centuries, consistently imposing a heavy burden on patients and healthcare systems worldwide due to their high incidence and recurrence rates. Advancements in endoscopy, imaging, genetics, molecular biology and bioinformatics have led to a deeper and more comprehensive understanding of the mechanism behind nephrolithiasis. Kidney stone formation is a complex, multi-step and long-term process involving the transformation of stone-forming salts from free ions into asymptomatic or symptomatic stones influenced by physical, chemical and biological factors. Among the various types of kidney stones observed in clinical practice, calcareous nephrolithiasis is currently the most common and exhibits the most intricate formation mechanism. Extensive research suggests that calcareous nephrolithiasis primarily originates from interstitial subepithelial calcified plaques and/or calcified blockages in the openings of collecting ducts. These calcified plaques and blockages eventually come into contact with urine in the renal pelvis, serving as a nidus for crystal formation and subsequent stone growth. Both pathways of stone formation share similar mechanisms, such as the drive of abnormal urine composition, involvement of oxidative stress and inflammation, and an imbalance of stone inhibitors and promoters. However, they also possess unique characteristics. Hence, this review aims to provide detailed description and present recent discoveries regarding the formation processes of calcareous nephrolithiasis from two distinct birthplaces: renal interstitium and tubule lumen.

Phagocytosis model of calcium oxalate monohydrate crystals generated using human induced pluripotent stem cell-derived macrophages.

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.

Tight junction and kidney stone disease.

Defects of tight junction (TJ) are involved in many diseases related to epithelial cell functions, including kidney stone disease (KSD), which is a common disease affecting humans for over a thousand years. This review provides brief overviews of KSD and TJ, and summarizes the knowledge on crystal-induced defects of TJ in renal tubular epithelial cells (RTECs) in KSD. Calcium oxalate (CaOx) crystals, particularly COM, disrupt TJ via p38 MAPK and ROS/Akt/p38 MAPK signaling pathways, filamentous actin (F-actin) reorganization and α-tubulin relocalization. Stabilizing p38 MAPK signaling, reactive oxygen species (ROS) production, F-actin and α-tubulin by using SB239063, N-acetyl-L-cysteine (NAC), phalloidin and docetaxel, respectively, successfully prevent the COM-induced TJ disruption and malfunction. Additionally, genetic disorders of renal TJ, including mutations and single nucleotide polymorphisms (SNPs) of CLDN2, CLDN10b, CLDN14, CLDN16 and CLDN19, also affect KSD. Finally, the role of TJ as a potential target for KSD therapeutics and prevention is also discussed.

STAT6 promoting oxalate crystal deposition-induced renal fibrosis by mediating macrophage-to-myofibroblast transition via inhibiting fatty acid oxidation.

OBJECTIVE AND DESIGN: Kidney stones commonly occur with a 50% recurrence rate within 5 years, and can elevate the risk of chronic kidney disease. Macrophage-to-myofibroblast transition (MMT) is a newly discovered mechanism that leads to progressive fibrosis in different forms of kidney disease. In this study, we aimed to investigate the role of MMT in renal fibrosis in glyoxylate-induced kidney stone mice and the mechanism by which signal transducer and activator of transcription 6 (STAT6) regulates MMT. METHODS: We collected non-functioning kidneys from patients with stones, established glyoxylate-induced calcium oxalate stone mice model and treated AS1517499 every other day in the treatment group, and constructed a STAT6-knockout RAW264.7 cell line. We first screened the enrichment pathway of the model by transcriptome sequencing; detected renal injury and fibrosis by hematoxylin eosin staining, Von Kossa staining and Sirius red staining; detected MMT levels by multiplexed immunofluorescence and flow cytometry; and verified the binding site of STAT6 at the PPARα promoter by chromatin immunoprecipitation. Fatty acid oxidation (FAO) and fibrosis-related genes were detected by western blot and real-time quantitative polymerase chain reaction. RESULTS: In this study, we found that FAO was downregulated, macrophages converted to myofibroblasts, and STAT6 expression was elevated in stone patients and glyoxylate-induced kidney stone mice. The promotion of FAO in macrophages attenuated MMT and upregulated fibrosis-related genes induced by calcium oxalate treatment. Further, inhibition of peroxisome proliferator-activated receptor-α (PPARα) eliminated the effect of STAT6 deletion on FAO and fibrosis-associated protein expression. Pharmacological inhibition of STAT6 also prevented the development of renal injury, lipid accumulation, MMT, and renal fibrosis. Mechanistically, STAT6 transcriptionally represses PPARα and FAO through cis-inducible elements located in the promoter region of the gene, thereby promoting MMT and renal fibrosis. CONCLUSIONS: These findings establish a role for STAT6 in kidney stone injury-induced renal fibrosis, and suggest that STAT6 may be a therapeutic target for progressive renal fibrosis in patients with nephrolithiasis.

Oxalate disrupts monocyte and macrophage cellular function via Interleukin-10 and mitochondrial reactive oxygen species (ROS) signaling.

Oxalate is a small compound found in certain plant-derived foods and is a major component of calcium oxalate (CaOx) kidney stones. Individuals that consume oxalate enriched meals have an increased risk of forming urinary crystals, which are precursors to CaOx kidney stones. We previously reported that a single dietary oxalate load induces nanocrystalluria and reduces monocyte cellular bioenergetics in healthy adults. The purpose of this study was to extend these investigations to identify specific oxalate-mediated mechanisms in monocytes and macrophages. We performed RNA-Sequencing analysis on monocytes isolated from healthy subjects exposed to a high oxalate (8 mmol) dietary load. RNA-sequencing revealed 1,198 genes were altered and Ingenuity Pathway Analysis demonstrated modifications in several pathways including Interleukin-10 (IL-10) anti-inflammatory cytokine signaling, mitochondrial metabolism and function, oxalic acid downstream signaling, and autophagy. Based on these findings, we hypothesized that oxalate induces mitochondrial and lysosomal dysfunction in monocytes and macrophages via IL-10 and reactive oxygen species (ROS) signaling which can be reversed with exogenous IL-10 or Mitoquinone (MitoQ; a mitochondrial targeted antioxidant). We exposed monocytes and macrophages to oxalate in an in-vitro setting which caused oxidative stress, a decline in IL-10 cytokine levels, mitochondrial and lysosomal dysfunction, and impaired autophagy in both cell types. Administration of exogenous IL-10 and MitoQ attenuated these responses. These findings suggest that oxalate impairs metabolism and immune response via IL-10 signaling and mitochondrial ROS generation in both monocytes and macrophages which can be potentially limited or reversed. Future studies will examine the benefits of these therapies on CaOx crystal formation and growth in vivo.

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.

Sel1-like proteins and peptides are the major Oxalobacter formigenes-derived factors stimulating oxalate transport by human intestinal epithelial cells.

Kidney stones (KSs) are very common, excruciating, and associated with tremendous healthcare cost, chronic kidney disease (CKD), and kidney failure (KF). Most KSs are composed of calcium oxalate and small increases in urinary oxalate concentration significantly enhance the stone risk. Oxalate also potentially contributes to CKD progression, kidney disease-associated cardiovascular diseases, and poor renal allograft survival. This emphasizes the urgent need for plasma and urinary oxalate lowering therapies, which can be achieved by enhancing enteric oxalate secretion. We previously identified Oxalobacter formigenes (O. formigenes)-derived factors secreted in its culture-conditioned medium (CM), which stimulate oxalate transport by human intestinal Caco2-BBE (C2) cells and reduce urinary oxalate excretion in hyperoxaluric mice by enhancing colonic oxalate secretion. Given their remarkable therapeutic potential, we now identified Sel1-like proteins as the major O. formigenes-derived secreted factors using mass spectrometry and functional assays. Crystal structures for six proteins were determined to confirm structures and better understand functions. OxBSel1-14-derived small peptides P8 and P9 were identified as the major factors, with P8 + 9 closely recapitulating the CM's effects, acting through the oxalate transporters SLC26A2 and SLC26A6 and PKA activation. Besides C2 cells, P8 + 9 also stimulate oxalate transport by human ileal and colonic organoids, confirming that they work in human tissues. In conclusion, P8 and P9 peptides are identified as the major O. formigenes-derived secreted factors and they have significant therapeutic potential for hyperoxalemia, hyperoxaluria, and related disorders, impacting the outcomes of patients suffering from KSs, enteric hyperoxaluria, primary hyperoxaluria, CKD, KF, and renal transplant recipients.NEW & NOTEWORTHY We previously identified Oxalobacter formigenes-derived secreted factors stimulating oxalate transport by human intestinal epithelial cells in vitro and reducing urinary oxalate excretion in hyperoxaluric mice by enhancing colonic oxalate secretion. We now identified Sel1-like proteins and small peptides as the major secreted factors and they have significant therapeutic potential for hyperoxalemia and hyperoxaluria, impacting the outcomes of patients suffering from kidney stones, primary and secondary hyperoxaluria, chronic kidney disease, kidney failure, and renal transplant recipients.

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.

Untargeted and targeted metabolomics reveal bile acid profile changes in rats with ethylene glycol-induced calcium oxalate nephrolithiasis.

Calcium oxalate (CaOx) nephrolithiasis is a prevalent disorder linked to metabolism. Examining metabolic alterations could potentially give an initial understanding of the origins of CaOx nephrolithiasis. This study aims to determine gut metabolic biomarkers differentiating CaOx nephrolithiasis utilizing untargeted and targeted metabolomics. CaOx nephrolithiasis model rats were built by 1% ethylene glycol administration. Histologic staining and renal function measurement revealed the presence of crystals in the lumen of the renal tubules, the renal injury and interstitial fibrosis in CaOx rats, demonstrating that the models of CaOx were established successfully. Hematoxylin & eosin (H&E) staining showed that CaOx group had inflammation and damage in the ileal tissue. Immunofluorescence and PCR results displayed that the tight junction proteins, ZO-1 and Occludin levels were decreased in the ileal tissues of the CaOx group. The untargeted metabolomic analysis revealed that 269 gut metabolites were differentially expressed between the CaOx group and the control group. Meanwhile, bile secretion, the main metabolic pathway in CaOx nephrolithiasis, was identified. Following, five significant bile acid metabolites were selected utilizing the targeted bile acid metabolomics, including Hyodeoxycholic acid (HDCA), Glycohyodeoxycholic acid (GHDCA), Nor-Deoxycholic Acid, omega-muricholic acid, and Taurolithocholic acid. Among these metabolites, HDCA and GHDCA presented the highest predictive accuracy with AUC = 1 to distinguish the CaOx group from the control group. As a result of network pharmacology, target genes of HDCA and GHDCA in CaOx nephrolithiasis were enriched in oxidative stress and apoptosis pathways. Conclusively, our study provides insight into bile acids metabolic changes related to CaOx nephrolithiasis. Although alterations in biochemical pathways indicate a complex pathology in CaOx rats, bile acid changes may serve as biomarkers of CaOx nephrolithiasis.

Increased renal uptake and urine excretion of oxidized LDL is possibly associated with formation of large calcium oxalate nephrolithiasis: a preliminary study.

PURPOSE: Growing evidence have suggested an association between nephrolithiasis and cardiovascular disease (CVD) with unclear mechanism. Oxidized low-density lipoproteins (oxLDL) induces atherosclerosis and was found to be the possible link between these two diseases. Our study aimed to examine the serum, urine and kidney expression of oxLDL in relation to large calcium oxalate (CaOx) renal stone disease. METHODS: A total of 67 large CaOx dominant renal stone patients and 31 stone-free controls were enrolled in the prospective case-control study. All participants were without known CVD history. Serum, urine, and kidney biopsy were collected before and during percutaneous nephrolithotomy, respectively. Enzyme-linked immunosorbent assays were used to assess serum and urine oxLDL, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), and high-sensitivity C-reactive protein (hsCRP). RESULTS: There was no significantly difference in circulating oxLDL, but serum hsCRP was significantly near two-fold higher in nephrolithiasis patients. Serum hsCRP was also correlated with stone maximal length. Urine oxLDL was significantly higher in the nephrolithiasis group and correlated with serum hsCRP and stone maximal length. Increased oxLDL uptake in kidney was found in nephrolithiasis patients, whereas no significantly renal expression of oxLDL was observed in controls. CONCLUSIONS: The renal uptake of oxLDL with increased oxLDL excretion from large CaOx renal stone formers, independent of increased circulating oxLDL, is a novel pathological finding in kidney stone disease and brings attention to the possible involvement of renal steatosis in the process of urolithiasis formation.

Ferroptosis in calcium oxalate kidney stone formation and the possible regulatory mechanism of ANKRD1.

The objective of this study was to explore the role of ferroptosis in the formation of calcium oxalate (CaOx) kidney stones and the regulatory mechanism of the ankyrin repeat domain 1 (ANKRD1) gene. The study found that the Nrf2/HO-1 and p53/SLC7A11 signaling pathways were activated in the kidney stone model group, and the expression of the ferroptosis marker proteins SLC7A11 and GPX4 was significantly reduced, while the expression of ACSL4 was significantly increased. The expression of the iron transport-related proteins CP and TF increased significantly, and Fe2+ accumulated in the cell. The expression of HMGB1 increased significantly. In addition, the level of intracellular oxidative stress was increased. The gene with the most significant difference caused by CaOx crystals in HK-2 cells was ANKRD1. Silencing or overexpression of ANKRD1 by lentiviral infection technology regulated the expression of the p53/SLC7A11 signaling pathway, which regulated the ferroptosis induced by CaOx crystals. In conclusion, CaOx crystals can mediate ferroptosis through the Nrf2/HO-1 and p53/SLC7A11 pathways, thereby weakening the resistance of HK-2 cells to oxidative stress and other unfavorable factors, enhancing cell damage, and increasing crystal adhesion and CaOx crystal deposition in the kidney. ANKRD1 participates in the formation and development of CaOx kidney stones by activating ferroptosis mediated by the p53/SLC7A11 pathway.

Meta-data analysis of kidney stone disease highlights ATP1A1 involvement in renal crystal formation.

Nephrolithiasis is a complicated disease affected by various environmental and genetic factors. Crystal-cell adhesion is a critical initiation process during kidney stone formation. However, genes regulated by environmental and genetic factors in this process remain unclear. In the present study, we integrated the gene expression profile data and the whole-exome sequencing data of patients with calcium stones, and found that ATP1A1 might be a key susceptibility gene involved in calcium stone formation. The study showed that the T-allele of rs11540947 in the 5'-untranslated region of ATP1A1 was associated with a higher risk of nephrolithiasis and lower activity of a promoter of ATP1A1. Calcium oxalate crystal deposition decreased ATP1A1 expression in vitro and in vivo and was accompanied by the activation of the ATP1A1/Src/ROS/p38/JNK/NF-κB signaling pathway. However, the overexpression of ATP1A1 or treatment with pNaKtide, a specific inhibitor of the ATP1A1/Src complex, inhibited the ATP1A1/Src signal system and alleviated oxidative stress, inflammatory responses, apoptosis, crystal-cell adhesion, and stone formation. Moreover, the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine reversed ATP1A1 down-regulation induced by crystal deposition. In conclusion, this is the first study to show that ATP1A1, a gene modulated by environmental factors and genetic variations, plays an important role in renal crystal formation, suggesting that ATP1A1 may be a potential therapeutic target for treating calcium stones.

Hyperoside Ameliorates Renal Tubular Oxidative Damage and Calcium Oxalate Deposition in Rats through AMPK/Nrf2 Signaling Axis.

Background: Nephrolithiasis is a common disease that seriously affects the health and life quality of patients. Despite the reported effect of hyperoside (Hyp) against nephrolithiasis, the specific mechanism has not been clarified. Therefore, this study is aimed at investigating the effect and potential mechanism of Hyp on renal injury and calcium oxalate (CaOx) crystal deposition. Methods: Rat and cell models of renal calculi were constructed by ethylene glycol (EG) and CaOx induction, respectively. The renal histopathological damage, CaOx crystal deposition, and renal function damage of rats were assessed by HE staining, Pizzolato staining, and biochemical detection of blood and urine parameters. MTT and crystal-cell adhesion assays were utilized to determine the activity of HK-2 cells and crystal adhesion ability, biochemical detection and enzyme-linked immunosorbent assay (ELISA) to measure the levels of oxidative stress-related substances and inflammatory factors, and western blot to test the expression levels of proteins related to the AMPK/Nrf2 signaling pathway. Results: Briefly speaking, Hyp could improve the renal histopathological injury and impaired renal function, reduce the deposition of CaOx crystals in the renal tissue of rats with renal calculi, and decrease the adhesion of crystals to CaOx-treated HK-2 cells. Besides, Hyp also significantly inhibited oxidative stress response. Furthermore, Hyp was associated with the downregulation of malondialdehyde, lactate dehydrogenase, and reactive oxygen species and upregulation of superoxide dismutase activity. Additionally, Hyp treatment also suppressed inflammatory response and had a correlation with declined levels of interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor. Further exploration of mechanism manifested that Hyp might play a protective role through promoting AMPK phosphorylation and nuclear translation of Nrf2 to activate the AMPK/Nrf2 signaling pathway. Conclusion: Hyp can improve renal pathological and functional damage, decrease CaOx crystal deposition, and inhibit oxidative stress and inflammatory response. Such effects may be achieved by activating the AMPK/Nrf2 signaling pathway.

LncRNA LINC01197 inhibited the formation of calcium oxalate-induced kidney stones by regulating miR-516b-5p/SIRT3/FOXO1 signaling pathway.

Long non-coding RNA (lncRNA) plays a key role in the regulation of calcium oxalate (CaOx) crystals-induced kidney stone formation and deposition. The purpose of this study is to study the effect of lncRNA LINC01197 on CaOx-induced kidney stone formation and the underlying mechanism. Crystal cell adhesion in HK-2 cells was evaluated by analyzing Ca2+ concentration. Apoptosis was detected by flow cytometry. The RT-qPCR and western blot were used to detect the mRNA and protein expression. Patients with kidneys stones showed down-regulated LINC01197 and SIRT3 expression, and up-regulated miR-516b-5p expression. LINC01197 knockdown promoted CaOx-induced cell adherence and cell apoptosis, increased Bax, decreased Bcl-2 expression. Luciferase reporter assay showed that SIRT3 expression was promoted by LINC01197 competing binds to miR-516b-5p. In addition, LINC01197 expression was promoted by SIRT3/FOXO1 overexpression, and could be reversed by FOXO1 knockdown. In conclusion, the present study revealed that lncRNA LINC01197 inhibited CaOx-induced kidney stones formation by regulating the miR-516b-5p/SIRT3/FOXO1 signaling pathway.

ePHex: a phase 3, double-blind, placebo-controlled, randomized study to evaluate long-term efficacy and safety of Oxalobacter formigenes in patients with primary hyperoxaluria.

BACKGROUND: Primary hyperoxalurias (PHs) are rare genetic diseases that increase the endogenous level of oxalate, a waste metabolite excreted predominantly by the kidneys and also the gut. Treatments aim to improve oxalate excretion, or reduce oxalate generation, to prevent kidney function deterioration. Oxalobacter formigenes is an oxalate metabolizing bacterium. This Phase III, double-blind, placebo-controlled randomized trial investigated the effectiveness of orally administered Oxabact™, a lyophilized O. formigenes formulation, at reducing plasma oxalate levels in patients suffering from PH. METHODS: Subjects (≥ 2 years of age) with a diagnosis of PH and maintained but suboptimal kidney function (mean estimated glomerular filtration rate at baseline < 90 mL/min/1.73 m2) were eligible to participate. Subjects were randomized to receive Oxabact or placebo twice daily for 52 weeks. Change from baseline in plasma oxalate concentration at Week 52 was the primary study endpoint. RESULTS: Forty-three subjects were screened, 25 were recruited and one was discontinued. At Week 52, O. formigenes was established in the gut of subjects receiving Oxabact. Despite decreasing plasma oxalate level in subjects treated with Oxabact, and stable/increased levels with placebo, there was no significant difference between groups in the primary outcome (Least Squares mean estimate of treatment difference was - 3.80 µmol/L; 95% CI: - 7.83, 0.23; p-value = 0.064). Kidney function remained stable in both treatments. CONCLUSIONS: Oxabact treatment may have stabilized/reduced plasma oxalate versus a rise with placebo, but the difference over 12 months was not statistically significant (p = 0.06). A subtle effect observed with Oxabact suggests that O. formigenes may aid in preventing kidney stones. A higher resolution version of the Graphical abstract is available as Supplementary information.

OLMALINC/OCT4/BMP2 axis enhances osteogenic-like phenotype of renal interstitial fibroblasts to participate in Randall's plaque formation.

BACKGROUND: Randall's plaques (RP) are identified as anchored sites for kidney calcium oxalate stones, but the mechanism remains unclear. Given the importance of osteogenic-like cells in RP formation and OCT4 in reprogramming differentiated cells to osteoblasts, the current study explored the potential role of OCT4 in RP formation. METHODS: OCT4 and biomineralization were evaluated in RP, and immunofluorescence co-staining was performed to identify these cells with alteration of OCT4 and osteogenic markers. Based on the analysis of tissue, we further investigated the mechanism of OCT4 in regulating osteogenic-like differentiation of primary human renal interstitial fibroblasts (hRIFs) in vitro and vivo. RESULTS: We identified the upregulated OCT4 in RP, with a positive correlation to osteogenic markers. Interestingly, fibroblast marker Vimentin was partially co-localized with upregulated OCT4 and osteogenic markers in RP. Further investigations revealed that OCT4 significantly enhanced the osteogenic-like phenotype of hRIFs in vitro and in vivo. Mechanically, OCT4 directly bound to BMP2 promoter and facilitated its CpG island demethylation to transcriptionally promote BMP2 expression. Furthermore, combination of RIP and RNA profiling uncovered that lncRNA OLMALINC physically interacted with OCT4 to promote its stabilization via disrupting the ubiquitination. Additionally, OLMALINC was upregulated in fibroblasts in RP visualized by FISH, and a positive correlation was revealed between OLMALINC and OCT4 in RP. CONCLUSIONS: The upregulation of OCT4 in hRIFs was a pathological feature of RP formation, and OLMALINC/OCT4/BMP2 axis facilitated hRIFs to acquire osteogenic-like phenotype under osteogenic conditions, through which the pathway might participate in RP formation. Our findings opened up a new avenue to better understand RP formation in which osteogenic-like process was partially triggered by lncRNAs and pluripotency maintenance related genes.