Renal Crystal Formation Research Articles

Dynamic change and preventive role of stress response via Keap1-Nrf2 during renal crystal formation.

Oxidative stress is a major risk factor for calcium oxalate nephrolithiasis. Reports suggest that oxidative stress response is induced in animals and humans with kidney stones. Keap1, Nrf2, and HO-1 are known as oxidative stress mediators. However, the association between oxidative stress response and stone formation is unclear. In this study, we analyzed oxidative stress response from the acute to the crystal formation phase when crystal formation was applied to renal crystal mice model and bioimaging mice and investigated the effect on crystal formation. In renal tissues, after glyoxylate administration, HO-1 increased for up to 6 h and returned to baseline at 24 h. This was observed following each daily dose until five days after the crystallization phase; however, the range of increase was attenuated. The possibility that Nrf2 activity influenced the number of crystals was considered in the experiment. Crystal formation increased in Nrf2-deficient mice and could be reduced by Nrf2 activators. In conclusion, the oxidative stress response via the Keap1-Nrf2 pathway may contribute to crystal formation. Particularly, this pathway may be a prospective target for drug development to prevent and cure nephrolithiasis.

Oxalate‑induced renal pyroptotic injury and crystal formation mediated by NLRP3‑GSDMD signaling invitro and invivo.

Calcium oxalate kidney stone has become an urgent issue due to its high incidence and recurrence rate. Thus, it is necessary to explore for mechanisms of calcium oxalate stones formation. Previous studies demonstrated that oxalate crystals could induce the activation of nucleotide‑binding domain and leucine‑rich repeat‑containing family pyrin domain‑containing 3 (NLRP3) inflammasome and change the renal tubular epithelium adhesion. However, the type and molecular mechanism of NLRP3 inflammasome‑mediated calcium oxalate stones formation still need to be further investigated. In the present study, it was confirmed that the NLRP3‑gasdermin D (GSDMD) signaling was involved in oxalate‑induced cell injury invitro and invivo. Inhibition of reactive oxygen species production could effectively prevent the NLRP3 inflammasome formation in oxalate‑treated HK‑2cells. NLRP3 gene silence could inhibit the DNA damage and cellular membrane injury of HK‑2cells treated with oxalate. The ultrastructural changes of several organelles and particular structures, similar to typical cell pyroptosis, were observed in oxalate‑stimulated HK‑2cells. NLRP3 gene silence could antagonize the oxalate‑induced injury and ultrastructure changes. Additionally, NSA (GSDMD inhibitor) could prevent the oxalate‑induced injury of membrane integrity in HK‑2cells. Moreover, oxalate crystals were significantly decreased in GSDMD‑/‑ mice compared with wild‑type mice with glyoxylic acid. Together, NLRP3‑GSDMD pathway was involved in the oxalate‑induced pyroptotic injury in HK‑2cells. GSDMD and its cleavage form GSDMD‑N played an important role in the oxalate‑induced renal cell injury and oxalate calcium crystals formation invitro and invivo. This provided a new target for prevention and treatment of oxalate nephropathy and oxalate calcium stones.

Primary hyperoxaluria I, II, III types in children (review of literature)

This review presents the latest data on the classification, pathogenesis, clinical and genetic features, and therapy of primary hyperoxaluria types I, II, and III in children with autosomal recessive inheritance. ORPHA portal of orphan diseases presents genes responsible for primary hyperoxaluria type I AGXT (93598); type II and type II GRHPR (93599), type III HOGA1 (93600). Worldwide genetic studies have established the pathogenesis, clinical phenotype and genotype features of primary hyperoxaluria. The pathogenesis of primary hyperoxaluria in children is based on impaired hepatic glyoxylate metabolism. The enzyme AGT catalyzes the conversion of L-alanine and glyoxylate to pyruvate and glycine, with vitamin B6 (pyridoxine) serving as a coenzyme for this reaction. Increased production of endogenous oxalate leads to increased blood oxalate concentrations and urinary oxalate excretion with the formation of renal calcium oxalate crystals and radiopaque concrements (calcium oxalate monohydrate – vevelite, calcium oxalate dihydrate – vedellite). High risk of progression to chronic kidney disease in primary hyperoxaluria in children of types I and II. Systemic oxalosis develops with increasing serum oxalate levels and the formation of calcium oxalate crystals with deposition in many organs and tissues. Therapy for primary hyperoxaluria in children includes: hydration (3l/m2/day) and citrates 100–150 mg/kg/day (potassium citrate 0.3–0.5 mmol/kg/day), pyridoxine at a dose of 5 to 20 mg/kg/day for vitamin B6 sensitive type I primary hyperoxaluria. Administration of oxalobacter formigenes and diet is effective. Combined liver and then kidney transplantation or simultaneous liver and kidney transplantation in patients with type I PH in B6-insensitive and isolated liver transplantation in B6-sensitive variants are performed. Timely molecular genetic testing in children with nephrocalcinosis makes it possible to establish a clinical and genetic diagnosis of type I, II, III PH, to carry out a personalised approach to treatment and to predict future health status.

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.

Hydroxyproline stimulates inflammation and reprograms macrophage signaling in a rat kidney stone model

Meals rich in oxalate are associated with calcium oxalate (CaOx) kidney stone disease. Hydroxy-L-proline (HLP) is an oxalate precursor found in milk and collagen-containing foods. HLP has been shown to induce CaOx crystal formation in rodents. The purpose of this study was to evaluate the effect of HLP induced oxalate levels on inflammation and renal leukocytes during crystal formation. Male Sprague-Dawley rats (6–8 weeks old) were fed a control diet containing no oxalate for 3 days before being randomized to continue the control diet or 5% HLP for up to 28 days. Blood, 24 h urine, and kidneys were collected on Days 0, 7, 14, or 28. Urinary oxalate levels, crystal deposition, and renal macrophage markers were evaluated using ion chromatography-mass spectrometry, immunohistochemistry, and qRT-PCR. Renal leukocytes were assessed using flow cytometry and RNA-sequencing. HLP feeding increased urinary oxalate levels and renal crystal formation in animals within 7 days. HLP also increased renal macrophage populations on Days 14 and 28. Transcriptome analysis revealed that renal macrophages from animals fed HLP for 7 days were involved in inflammatory response and disease, stress response to LPS, oxidative stress, and immune cell trafficking. Renal macrophages isolated on Day 14 were involved in cell-mediated immunological pathways, ion homeostasis, and inflammatory response. Collectively, these findings suggest that HLP-mediated oxalate levels induce markers of inflammation, leukocyte populations, and reprograms signaling pathways in macrophages in a time-dependent manner. Additional studies investigating the significance of oxalate on renal macrophages could aid in our understanding of kidney stone formation.

Bioinformatics analysis of pathways of renal infiltrating macrophages in different renal disease models.

BackgroundRecent studies have suggested that macrophages are significantly involved in different renal diseases. However, the role of these renal infiltrating macrophages has not been entirely uncovered. To further clarify the underlying mechanism and identify therapeutic targets, a bioinformatic analysis based on transcriptome profiles was performed.MethodsThree transcription profiling datasets, GSE27045, GSE51466 and GSE75808, were obtained from the Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) were assessed by Gene Ontology (GO) functional annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and gene set enrichment analysis (GSEA).ResultsThe classic signaling pathways and metabolic pathways of macrophages infiltrating the kidney in different pathophysiological processes, including lupus nephritis (LN), renal crystal formation and renal ischemia-reperfusion injury (IRI), were analysed. Furthermore, the common classical pathways significantly altered in the three renal disorders were the oxidative phosphorylation, VEGF signaling and JAK/STAT signaling pathways, while the renin-angiotensin system was uniquely altered in LN, the glycolysis and gluconeogenesis pathways were uniquely altered in models of renal crystal formation, and the calcium signaling pathway was specific to renal IRI.ConclusionsVia bioinformatics analysis, this study revealed the transcriptional features of macrophages in murine LN, renal crystal formation and IRI models, which may serve as promising targets for mechanistic research and the clinical treatment of multiple renal diseases.

Short-Chain Fatty Acids Reduced Renal Calcium Oxalate Stones by Regulating the Expression of Intestinal Oxalate Transporter SLC26A6.

ABSTRACTRenal calcium oxalate (CaOx) stone is a common urologic disease with a high prevalence and recurrence rate. However, short-chain fatty acids (SCFAs) are less often reported in the prevention of urolithiasis. This study aimed to explore the effect of SCFAs on the renal CaOx stone formation and the underlying mechanisms. Ethylene glycol was used to induce renal CaOx crystals in rats. SCFAs (acetate, propionate, or butyrate) were added as supplements to the drinking water with or without antibiotics. Because intestinal oxalate transporters SLC26A6 and SLC26A3 regulate the excretion and absorption of oxalate in the intestine, we injected adeno-associated virus 9 (AAV9)-SLC26A6-shRNA (short hairpin RNA) and AAV9-SLC26A3 into the tail vein of rats to suppress SLC26A6 and overexpress SLC26A3 expression in the intestine, respectively, to explore the role of SLC26A3 and SLC26A6 (SLC26A3/6) in the reduction of renal CaOx crystals induced by SCFAs. Results showed that SCFAs reduced renal CaOx crystals and urinary oxalate levels but, however, increased the abundance of SCFA-producing bacteria and cecum SCFA levels. SCFA supplements still reduced renal crystals and urinary oxalate after gut microbiota depletion. Propionate and butyrate downregulated intestinal oxalate transporter SLC26A3 expression, while acetate and propionate upregulated SLC26A6 expression, both in vivo and in vitro. AAV9-SLC26A3 exerted a protective effect against renal crystals, while AAV9-SLC26A6-shRNA contributed to the renal crystal formation even though the SCFAs were supplemented. In conclusion, SCFAs could reduce urinary oxalate and renal CaOx stones through the oxalate transporter SLC26A6 in the intestine. SCFAs may be new supplements for preventing the formation of renal CaOx stones.IMPORTANCE Some studies found that the relative abundances of short-chain-fatty-acid (SCFA)-producing bacteria were lower in the gut microbiota of renal stone patients than healthy controls. Our previous study demonstrated that SCFAs could reduce the formation of renal calcium oxalate (CaOx) stones, but the mechanism is still unknown. In this study, we found that SCFAs (acetate, propionate, and butyrate) reduced the formation of renal calcium oxalate (CaOx) crystals and the level of urinary oxalate. Depleting gut microbiota increased the amount of renal crystals in model rats, and SCFA supplements reduced renal crystals and urinary oxalate after gut microbiota depletion. Intestinal oxalate transporter SLC26A6 was a direct target of SCFAs. Our findings suggested that SCFAs could reduce urinary oxalate and renal CaOx stones through the oxalate transporter SLC26A6 in the intestine. SCFAs may be new supplements for preventing the formation of renal CaOx stones.

Recent advances on the mechanisms of kidney stone formation (Review).

Kidney stone disease is one of the oldest diseases known to medicine; however, the mechanisms of stone formation and development remain largely unclear. Over the past decades, a variety of theories and strategies have been developed and utilized in the surgical management of kidney stones, as a result of recent technological advances. Observations from the authors and other research groups suggest that there are five entirely different main mechanisms for kidney stone formation. Urinary supersaturation and crystallization are the driving force for intrarenal crystal precipitation. Randall's plaques are recognized as the origin of calcium oxalate stone formation. Sex hormones may be key players in the development of nephrolithiasis and may thus be potential targets for new drugs to suppress kidney stone formation. The microbiome, including urease-producing bacteria, nanobacteria and intestinal microbiota, is likely to have a profound effect on urological health, both positive and negative, owing to its metabolic output and other contributions. Lastly, the immune response, and particularly macrophage differentiation, play crucial roles in renal calcium oxalate crystal formation. In the present study, the current knowledge for each of these five aspects of kidney stone formation is reviewed. This knowledge may be used to explore novel research opportunities and improve the understanding of the initiation and development of kidney stones for urologists, nephrologists and primary care.

Essential roles of oncostatin M receptor \u03b2 signaling in renal crystal formation in mice

Oncostatin M (OSM), a member of the IL-6 family of cytokines, has important roles in renal diseases. The relationship between OSM and kidney stone disease, however, remains unclear. To investigate the roles of OSM in the development of kidney stone disease, we generated a mouse model of renal crystal formation using OSM receptor β (OSMRβ)-deficient mice (OSMRβ−/− mice). There were fewer renal crystal deposits in OSMRβ−/− mice than in wild-type (WT) mice. Crystal-binding molecules (osteopontin, annexin A1, and annexin A2), inflammatory cytokines (TNF-α and IL-1β), and fibrosis markers (TGF-β, collagen 1a2, and α-smooth muscle actin) were also decreased in the kidneys of OSMRβ−/− mice compared with those in WT mice. Immunofluorescence staining showed that OSMRβ was expressed in renal tubular epithelial cells (RTECs) and renal fibroblasts in the model of renal crystal formation. In the cultured RTECs and renal fibroblasts, OSM directly induced the expression of crystal-binding molecules and fibrosis markers. Expressions of inflammatory cytokines were increased by stimulation with OSM in cultured renal fibroblasts. OSM may promote the formation of renal crystal deposits by directly acting on RTECs and renal fibroblasts to produce crystal-binding molecules and inflammatory cytokines.

The relationship between gut microbiota and short chain fatty acids in the renal calcium oxalate stones disease.

The relationship of gut microbiota and calcium oxalate stone has been limited investigated, especially with no study of gut microbiota and short chain fatty acids (SCFAs) in nephrolithiasis. We provided Sprague Dawley rats of renal calcium oxalate stones with antibiotics and examined the renal crystals deposition. We also performed a case-control study by analyzing 16S rRNA microbial profiling, shotgun metagenomics and SCFAs in 153 fecal samples from non-kidney stone (NS) controls, patients with occasional renal calcium oxalate stones (OS) and patients with recurrent stones (RS). Antibiotics reduced bacterial load in feces and could promote the formation of renal calcium crystals in model rats. In addition, both OS and RS patients exhibited higher fecal microbial diversity than NS controls. Several SCFAs-producing gut bacteria, as well as metabolic pathways associated with SCFAs production, were considerably lower in the gut microbiota among the kidney stone patients compared with the NS controls. Representation of genes involved in oxalate degradation showed no significance difference among groups. However, fecal acetic acid concentration was the highest in RS patients with high level of urinary oxalate, which was positively correlated with genes involvement in oxalate synthesis. Administration of SCFAs reduced renal crystals. These results shed new light on bacteria and SCFAs, which may promote the development of treatment strategy in nephrolithiasis.

PD04-09 BROWN ADIPOCYTES SUPPRESS KIDNEY STONE FORMATION VIA HEAT-PRODUCING PROTEIN, UNCOUPLING PROTEIN 1

You have accessJournal of UrologyStone Disease: Basic Research & Pathophysiology I (PD04)1 Apr 2020PD04-09 BROWN ADIPOCYTES SUPPRESS KIDNEY STONE FORMATION VIA HEAT-PRODUCING PROTEIN, UNCOUPLING PROTEIN 1 Teruaki Sugino*, Yutaro Tanaka, Rei Unno, Kazumi Taguchi, Shuzo Hamamoto, Ryosuke Ando, Atsushi Okada, Tohru Mogami, Hitoshi Yamashita, and Takahiro Yasui Teruaki Sugino*Teruaki Sugino* More articles by this author , Yutaro TanakaYutaro Tanaka More articles by this author , Rei UnnoRei Unno More articles by this author , Kazumi TaguchiKazumi Taguchi More articles by this author , Shuzo HamamotoShuzo Hamamoto More articles by this author , Ryosuke AndoRyosuke Ando More articles by this author , Atsushi OkadaAtsushi Okada More articles by this author , Tohru MogamiTohru Mogami More articles by this author , Hitoshi YamashitaHitoshi Yamashita More articles by this author , and Takahiro YasuiTakahiro Yasui More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000824.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Our recent studies have revealed that adipocytokine secreted by adipocytes are important for the formation of kidney stones. Brown adipocytes are reported to suppress inflammatory adipocytokine expression via a specific protein—uncoupling protein 1 (UCP1). In this study, we investigated the effect of brown adipocytes and UCP1 on kidney stone formation. METHODS: Study 1: Nine-week-old male C57BL/6 mice underwent sham or brown adipose tissue transplant surgery (sham group, transplant group, n = 6 in each group). Three weeks after surgery, renal crystal deposits were introduced via daily intra-abdominal injections of 80 mg/kg glyoxylate (GOX). Study 2: Renal crystal deposits were introduced via daily intra-abdominal injections of 80 mg/kg GOX in wild-type and defective type 8-week-old male UCP1-deficient mice (B6.129-Ucp1tm1Kz/J) (WT group, KO group, n = 6 in each group). Fat tissues, kidneys, and 24-hour urine samples of the mice were collected in both studies. RESULTS: Study 1: In the transplant group, the amount of renal crystal deposits was significantly lower (3.28 times) than that in the sham group (Fig. 1a), and lower expression of inflammatory markers, such as chemokine (C-C motif) ligand 2 (Ccl2), EGF module-containing mucin-like receptor 1 (Emr1), tumor necrosis factor (Tnf), and secreted phosphoprotein 1 (Spp1)was observed in the kidney. Inductive brown adipocytes were observed in the perirenal fat tissue in the transplant group. Study 2: In the KO group, the amount of renal crystal deposits was significantly higher (3.28 times) than that in the WT group (Fig. 1b), and higher expression of Emr1, Tnf, and Spp1 was observed in the kidney. Infiltration of inflammatory cells was strongly observed in brown adipose tissue in the KO group. There were no significant differences in the 24-hour urine samples throughout the two studies. CONCLUSIONS: Brown adipose tissue transplantation suppressed renal crystal formation via anti-inflammatory effects, and UCP1 would be necessary in this mechanism. Brown adipocytes can be the therapeutic target for kidney stone formation. Source of Funding: none © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e81-e82 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Teruaki Sugino* More articles by this author Yutaro Tanaka More articles by this author Rei Unno More articles by this author Kazumi Taguchi More articles by this author Shuzo Hamamoto More articles by this author Ryosuke Ando More articles by this author Atsushi Okada More articles by this author Tohru Mogami More articles by this author Hitoshi Yamashita More articles by this author Takahiro Yasui More articles by this author Expand All Advertisement PDF downloadLoading ...

The Relationship between Gut Microbiota and Short Chain Fatty Acids in the Renal Calcium Oxalate Stones Disease

Background: The relationship of gut microbiota and calcium oxalate stone has been limited investigated, especially with no study of gut microbiota and short chain fatty acids (SCFAs) in nephrolithiasis. Methods: We provided Sprague Dawley rats of renal calcium oxalate stones with antibiotics and examined the renal crystals deposition. We also performed a case-control study by analyzing 16S rRNA microbial profiling, shotgun metagenomics and SCFAs in 153 fecal samples from non-kidney stone (NS) controls, patients with occasional renal calcium oxalate stones (OS) and patients with recurrent stones (RS). Findings: Antibiotics reduced bacterial load in feces and could promote the formation of renal calcium crystals in model rats. In addition, both OS and RS patients exhibited higher fecal microbial diversity than NS controls. Several SCFAs-producing gut bacteria, as well as metabolic pathways associated with SCFAs production, were considerably lower in the gut microbiota among the kidney stone patients compared with the NS controls. Representation of genes involved in oxalate degradation showed no significance difference among groups. However, fecal acetic acid concentration was the highest in RS patients with high level of urinary oxalate, which was positively correlated with genes involvement in oxalate synthesis. Interpretation: Depletion of gut microbiota promoted the formation of renal crystals. Fecal SCFAs dominant patients, with altered gut microbiota composition and the functional metagenome, were associated with oxalate synthesis. Funding Statement: This work was supported by the National Natural Science Foundation of China [81770703, 81970602]; the Foundation of Science & Technology Department of Sichuan Province [2018SZ0118]; and the 1·3·5 Project for Disciplines of Excellence, West China Hospital, Sichuan University [ZYGD18011, ZYJC18015, ZY2016104]. Declaration of Interests: The authors declare that they have no conflicts of interest. Ethics Approval Statement: The animal experiment was performed in accordance with the policies of the West China Hospital of Sichuan University Medical Research Ethics Committee (2017063A). This study was approved by the West China Hospital of Sichuan University Medical Research Ethics Committee (2018182), and informed consents were obtained from each participant.

Dietary vinegar prevents kidney stone recurrence via epigenetic regulations.

BackgroundEpidemiological evidence of over 9000 people suggests that daily intake of vinegar whose principal bioactive component is acetic acid is associated with a reduced risk of nephrolithiasis. The underlying mechanism, however, remains largely unknown.MethodsWe examined the in vitro and in vivo anti-nephrolithiasis effects of vinegar and acetate. A randomized study was performed to confirm the effects of vinegar in humans.FindingsWe found individuals with daily consumption of vinegar compared to those without have a higher citrate and a lower calcium excretion in urine, two critical molecules for calcium oxalate (CaOx) kidney stone in humans. We observed that oral administration of vinegar or 5% acetate increased citrate and reduced calcium in urinary excretion, and finally suppressed renal CaOx crystal formation in a rat model. Mechanism dissection suggested that acetate enhanced acetylation of Histone H3 in renal tubular cells and promoted expression of microRNAs-130a-3p, −148b-3p and -374b-5p by increasing H3K9, H3K27 acetylation at their promoter regions. These miRNAs can suppress the expression of Nadc1 and Cldn14, thus enhancing urinary citrate excretion and reducing urinary calcium excretion. Significantly these mechanistic findings were confirmed in human kidney tissues, suggesting similar mechanistic relationships exist in humans. Results from a pilot clinical study indicated that daily intake of vinegar reduced stone recurrence, increased citrate and reduced calcium in urinary excretion in CaOx stone formers without adverse side effects.InterpretationVinegar prevents renal CaOx crystal formation through influencing urinary citrate and calcium excretion via epigenetic regulations. Vinegar consumption is a promising strategy to prevent CaOx nephrolithiasis occurrence and recurrence.FundNational Natural Science Foundations of China and National Natural Science Foundation of Guangdong Province.

Pioglitazone decreased renal calcium oxalate crystal formation by suppressing M1 macrophage polarization via the PPAR-γ-miR-23 axis.

Interaction of pioglitazone (PGZ) and macrophages (Mps) in renal crystal formation remains unclear. In the present study, we investigated the possible mechanisms involved with Mps of PGZ in suppressing renal crystal formation. Crystal formation in the mouse kidney was detected using polarized light optical microscopy and Pizzolato staining. Gene expression was detected by Western blot analysis, quantitative RT-PCR, immunohistochemistry, immunofluorescence, and ELISA. Mp phenotypes were identified by flow cytometric analysis. Cell apoptosis was detected with TUNEL assay, and tubular injury was detected with periodic acid-Schiff staining. Interaction of peroxisome proliferator-activated receptor (PPAR)-γ and promoter was determined by chromatin immunoprecipitation assay. Luciferase reporter assay was performed to authenticate target genes of miRNA-23 (miR-23). Recombinant adenovirus was used to elucidate the role of miR-23 in vivo. Renal crystal formation, inflammation, tubular injury, and cell apoptosis were significantly marked in glyoxylic acid-treated groups and significantly decreased in PGZ-treated groups. PGZ significantly reduced Mp infiltration and M1 Mp polarization in the kidney. In vitro, PGZ shifted crystal-stimulated M1-predominant Mps to M2-predominant Mps, which were anti-inflammatory. PPAR-γ could directly bind to one PPAR-γ regulatory element in the promoter of pre-miR-23 to promote expression of miR-23 in Mps. We identified two downstream target genes of miR-23, interferon regulatory factor 1 and Pknox1. Moreover, miR-23 decreased crystal deposition, M1 Mp polarization, and injury in the kidney. This study has proven that PGZ decreased renal calcium oxalate crystal formation and renal inflammatory injury by suppressing M1 Mp polarization through a PPAR-γ-miR-23-interferon regulatory factor 1/Pknox1 axis. PGZ is liable to be a potential therapeutic medicine for treating urolithiasis.

Re: Sirtuin 3 Suppresses the Formation of Renal Calcium Oxalate Crystals through Promoting M2 Polarization of Macrophages

Re: Sirtuin 3 Suppresses the Formation of Renal Calcium Oxalate Crystals through Promoting M2 Polarization of Macrophages

Brown adipocytes and β3-stimulant-induced brown-like adipocytes contribute to the prevention of renal crystal formation.

According to recent studies, kidney stones are associated with metabolic syndrome. We focused on brown adipocytes and β3-stimulant-induced brown-like adipocytes to investigate how these adipocytes influence kidney stone disease. For the interscapular brown adipose tissue (iBAT) removal experiment, mice were subjected to either iBAT removal or sham operation (X-BAT group or sham group), and, after 3 wk, renal crystal deposition was induced by intra-abdominal injection of glyoxylate (GOX) for 6 days. For the β3-stimulant experiment, mice were administered intra-abdominal injections of the β3-stimulant (β3-group) or saline (control group) for 6 days. Thereafter, renal crystal deposition was induced by intra-abdominal injection of GOX for 6 days. iBAT removal decreased the expression of Sod1 and increased that of chemokine (C-C motif) ligand 2 (Ccl2), EGF module-containing mucin-like receptor 1 (Emr1), and tumor necrosis factor (Tnf) in the kidneys. Renal crystal deposition was 2.06-fold higher in the X-BAT group than in the sham group. The β3-stimulant caused differentiation of white adipocytes into brown-like adipocytes. In the kidneys of the β3-group, the expression of Ccl2 and Emr1 decreased and that of Sod1 increased. Renal crystal deposition was 0.17-fold lower in the β3-group than in the control group. In summary, iBAT removal promoted kidney inflammation and renal crystal formation. β3-Stimulant-induced brown-like adipocytes reduced inflammation and improved antioxidant action in the kidneys, which suppressed renal crystal formation. This is the first report on the therapeutic role of brown and brown-like adipocytes for kidney stone formation.

199 - β3 Stimulant contributes to the prevention of renal crystal formation via differentiation of beige adipocytes

199 - β3 Stimulant contributes to the prevention of renal crystal formation via differentiation of beige adipocytes

Protective effect of salvianolic acid B against oxidative injury associated with cystine stone formation

The aim of this study was to investigate the role of oxidative stress in cystine crystal formation and whether salvianolic acid B, a natural antioxidant, could prevent cystine-mediated oxidative injury in vivo and in vitro. The levels of oxidative stress and antioxidase activity in cystine stone patients were assessed. Then, the oxidative stress exerted by cystine on human kidney-2 (HK-2) cell viability and biochemical parameters including antioxidase activity and antioxidant protein expression were evaluated, and the protective action of salvianolic acid B was also examined. Finally, salvianolic acid B was tested to determine whether it could prevent or reduce renal crystal formation in Slc7a9 knockout mice. The activity levels of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were decreased, and the amount of malondialdehyde (MDA) was increased in patients with cystine stones compared with people without cystine stones (p < 0.05). Significant reductions in cell viability, antioxidase activity and antioxidant protein expression levels were found in the cystine group compared with controls. However, such oxidative injuries were prevented by salvianolic acid B. In the animal study, loose crystals with white spots were seen in the renal parenchyma, bilateral renal pelvis and bladders in the Slc7a9 knockout group. In contrast, no renal crystals were seen in the control group, and markedly fewer crystals with significantly higher antioxidase activity and diminished oxidative stress were detected in the salvianolic acid B group. Cystine cytotoxicity in vitro and cystine stone formation in vivo were associated with oxidative stress, and salvianolic acid B could protect against cystine stone-induced injury.

Metformin prevents nephrolithiasis formation by inhibiting the expression of OPN and MCP-1 invitro and invivo.

Treatment targeting osteopontin (OPN) and monocyte chemoattractant protein 1 (MCP-1) has been recognized as a novel approach in renal crystal formation. The present study was designed to investigate the suppressive effects of metformin on nephrolithiasis formation and its potential mechanism. The cytotoxicity of metformin on MDCK and HK-2 cells was determined using a Cell Counting Kit-8 assay in vitro. Subsequently, the mRNA transcription and protein expression levels of MCP-1 and OPN were detected by reverse transcription-quantitative-polymerase chain reaction analysis, western blot analysis and ELISA. Male Sprague-Dawley rats were divided into a control group, ethylene glycol (EG) group and EG + metformin group. The expression levels of MCP-1 and OPN and crystal formations were evaluated in renal tissues following an 8-week treatment period. In vitro, metformin significantly inhibited the production of MCP-1 and OPN induced by oxalate at the mRNA and protein expression levels. In vivo, increased expression levels of MCP-1 and OPN were detected in the EG group compared with the controls, and this upregulation was reversed in the EG + metformin group. Renal crystal deposition in the EG + metformin group was markedly decreased compared with that in the EG group. Therefore, the results of the study suggest that metformin suppressed urinary crystal deposit formation, possibly by mediating the expression of inflammatory mediators OPN and MCP-1.

Vitamin K1 Inhibition of Renal Crystal Formation through Matrix Gla Protein in the Kidney

Background and Objectives: Vitamin K (VK) plays a major role in modifying the binding of calcium in bones and blood vessels. Understanding the effect of VK on crystal formation in the kidney would contribute to advancing the treatment and prevention of kidney stones. Methods: Rats were treated with vitamin K1 (VK1) for 8 weeks. VK1 levels were detected and crystal formation were observed. HK2 cells were exposed to calcium oxalate monohydrate crystals. Apoptosis and cell viability were detected. Crystal deposition was analyzed using atomic absorption assay. The adenovirus vectors expressing matrix Gla protein (MGP) and siMGP were constructed to elucidate the effect and mechanism of VK1 on crystal formation. MGP expression in vivo and in vitro was analyzed by Western blot. The mRNA levels of monocyte chemoattractant protein-1 (MCP-1) and collagen I was measured by semiquantitative RT-PCR. Results: The concentrations of VK1 in whole blood and kidney tissues rose under treatment with VK1. Crystal formation was inhibited from the second to the 6th week, the frequency and quality of crystal formation decreased significantly, and the location of crystal formation was limited to a greater extent in the rats treated by VK1 compared to the control group. Warfarin treatment in the crystals-exposed HK2 cells significantly increased the number of crystals adhering to cells and the number of apoptotic cells and reduced cell viability. VK1 treatment reversed warfarin’s above influence. VK1 inhibited the upregulations of MCP-1 and collagen I in kidney tissues under crystal load. VK1 treatment increased MGP expression in vivo and in vitro, and MGP is necessary for VK1 to play a role in crystal deposition in cells. Conclusions: VK1 treatment can inhibit the formation of renal crystals in vivo. VK1 increases MGP expression and functions through MGP to reduce crystal deposition in cells and provide cell protection. Our findings suggest that VK1 treatment could be a potential strategy for the treatment and prevention of nephrolithiasis.