Renal Calcium Oxalate Research Articles

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.

Effect of dimethyl fumarate on the renal calcium oxalate stone formation in rats and possible mechanism

Objective To observe the effect of dimethyl fumarate (DMF) on the renal calcium oxalate crystals formation in SD rats induced by glyoxylic acid monohydrate, and explore the possible mechanism. Methods Thirty male Sprague-Dawley rats were randomly divided into 3 groups: control group, model group and DMF group (n=10 each). The control group was free to drink purified water. Model group was induced by intraperitoneal injection of glyoxylic acid monohydrate to establish a kidney calcium oxalate stone model, and DMF group was treat with DMF by gastric administration on the basis of modeling. After 10 days of continuous treatment, the 24 h urine, serum and kidney of rats in each group were collected. Serum creatinine (SCr) value, calcium and oxalate (Ox) contents in urine were measured. Von Kossa staining method was used to detect the deposition of calcium oxalate crystals in kidney tissue. Western blotting was used to detect the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and HO-1 protein in renal tissue. The frozen section DHE staining was used to detect the ROS level in the kidney, thiobarbituric acid method was used to test malondialdehyde (MDA) content, and the xanthine oxidase method was used to detect superoxide dismutase (SOD) activity. Results There was no visible calcium crystal in the kidney of the control group. Compared to the model group, the amounts of renal calcium crystal were significantly reduced in the DMF group (76.07±4.87) /HP vs. (37.94±4.63) /HP (t=17.940, P 0.05). There was no significant difference in 24-hour urinary calcium ion content between the three groups (F=1.670, P>0.05). Compared with the control group, renal ROS levels (t=20.920, P<0.01), Nrf2 (t=17.290, P<0.01) and HO-1 protein (t=38.030, P<0.01) expression levels were significantly increased in the model group. Compared with the model group, renal ROS levels were significantly reduced in the DMF group (t=16.000, P<0.01), while the expression levels of Nrf2 (t=25.580, P<0.01) and HO-1 protein (t=79.290, P<0.01) were significantly increased. Conclusion The formation of calcium oxalate kidney stones is related to oxalate-mediated oxidative stress. DMF may interfere with the pathogenesis of calcium oxalate kidney stones by activating Nrf2 to induce HO-1 to inhibit oxidative stress. Key words: Calcium oxalate stones; Oxidative stress; Nuclear factor erythroid 2-related factor 2; Dimethyl fumarate

Increased Expression of Inflammatory Genes Using RNAseq in Kidney Tissue in Felines with Renal Disease or Calcium Oxalate Stones (P09-005-19)

ObjectivesInflammatory processes play an important role in the initiation and progression of kidney disease. Renal calcium oxalate stone formed from the crystal deposits in the tubular epithelial cells can increase reactive oxygen species and subsequent inflammation. We investigated the gene expression MethodsAt the time of death the circulating levels of creatinine and SDMA, markers of kidney decline, were significantly higher in cats with renal disease (n = 11) or stone-forming cats (CaOx, n = 12) when compared to controls (n = 19). ResultsWe identified 3 candidate genes including complement C3d receptor (CR2), MMP-7, and IKAROS family zinc finger 3, that were maximally up-regulated in renal disease with fold increases of 14.3, 13.9, and 10.9 when compared to controls (p < 0.01). Other selected inflammatory markers that were significantly increased but less than 10-fold included IL7R, TLR8, CXCR4, LCP1, IL2RB, and MSR1. The profile for maximally up-regulated genes in CaOx cats was somewhat similar when compared with those with renal disease with the exception of MMP-7. In CaOx cats the maximal up-regulation occurred in CR2, IKAROS family zinc finger 3, and L-selectin with fold increases of 33.9, 16.8, and 12.6 when compared to controls (p < 0.01). Selected inflammatory markers that were significantly increased, but less than 10-fold, included CD37, DOCK2, LCP1, CCL19, and TLR7 in CaOx compared to controls. Interestingly, there was a significant increase in Fc fragment of IgM receptor (FCMR) in both renal disease (12.4 fold) and CaOx (13.2 fold) when compared to controls. Our results identify newer immune biomarkers that are novel targets for attenuating kidney dysfunction and while there are similarities in the inflammatory genes upregulated in cats with kidney disease and stone formers, the expression profile of upregulated genes, taken as a whole, is different in both groups. ConclusionsThese results indicate that there are somewhat different renal responses to calcium oxalate stones and renal disease which suggests the optimal anti-inflammatory nutritional therapy may be different. Funding SourcesHill’s Pet Nutrition, Inc.

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

The Protective Roles of Estrogen Receptor β in Renal Calcium Oxalate Crystal Formation via Reducing the Liver Oxalate Biosynthesis and Renal Oxidative Stress-Mediated Cell Injury.

Females develop kidney stones less frequently than males do. However, it is unclear if this gender difference is related to altered estrogen/estrogen receptor (ER) signaling. Here, we found that ER beta (ERβ) signals could suppress hepatic oxalate biosynthesis via transcriptional upregulation of the glyoxylate aminotransferase (AGT1) expression. Results from multiple in vitro renal cell lines also found that ERβ could function via suppressing the oxalate-induced injury through increasing the reactive oxygen species (ROS) production that led to a decrease of the renal calcium oxalate (CaOx) crystal deposition. Mechanism study results showed that ERβ suppressed oxalate-induced oxidative stress via transcriptional suppression of the NADPH oxidase subunit 2 (NOX2) through direct binding to the estrogen response elements (EREs) on the NOX2 5′ promoter. We further applied two in vivo mouse models with glyoxylate-induced renal CaOx crystal deposition and one rat model with 5% hydroxyl-L-proline-induced renal CaOx crystal deposition. Our data demonstrated that mice lacking ERβ (ERβKO) as well as mice or rats treated with ERβ antagonist PHTPP had increased renal CaOx crystal deposition with increased urinary oxalate excretion and renal ROS production. Importantly, targeting ERβ-regulated NOX2 with the NADPH oxidase inhibitor, apocynin, can suppress the renal CaOx crystal deposition in the in vivo mouse model. Together, results from multiple in vitro cell lines and in vivo mouse/rat models all demonstrate that ERβ may protect against renal CaOx crystal deposition via inhibiting the hepatic oxalate biosynthesis and oxidative stress-induced renal injury.

208 - The role of gut microbiome and short chain fatty acids in renal calcium oxalate stones formation

208 - The role of gut microbiome and short chain fatty acids in renal calcium oxalate stones formation

Active Phagocytosis and Diachronic Processing of Calcium Oxalate Monohydrate Crystals in an in vitro Macrophage Model

Background: We previously discovered that renal macrophages (Mφs) phagocytose renal calcium oxalate monohydrate (COM) crystals. This study investigated the processing of engulfed crystals using in vitro models. Methods: J774.1 mouse Mφs were exposed to COM crystals and observed for 24 h using polarized light microscopy with/without cytochalasin B (CB), an inhibitor of phagocytosis, to confirm active crystal phagocytosis. LysoTracker and immunohistochemical staining using transmission electron microscopy for lysosomal-associated membrane protein 1 were used to confirm engulfed COM crystal uptake into lysosomes. Diachronic tracking of specific Mφs was performed to capture the entire course of engulfed COM crystal processing using polarized light microscopy. Follow-up studies of fluorescent COM (f-COM) crystals using imaging cytometry were performed in the presence and absence of nigericin to dissipate the pH gradient in acidic organelles. Results: Phagocytosis rates increased with COM density and were significantly lower in cells treated with CB (p < 0.01). We observed that engulfed crystals colocalized within lysosomes of the Mφs; moreover, diachronic observation indicated that the engulfed COM crystals were subdivided during Mφ division and eliminated by the 7th day of culture. Additionally, imaging cytometry showed that the fluorescence level of f-COM crystals in the nigericin (–) group after 48 h was significantly lower than that in the nigericin (+) group. Conclusions: This study confirmed active phagocytosis and lysosomal processing of engulfed COM crystals by Mφs. This discovery is expected to contribute to the development of future drugs that enhance the COM crystal phagocytic ability of Mφs.

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

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

Allicin attenuates calcium oxalate crystal deposition in the rat kidney by regulating gap junction function.

Renal calculus is a global common urological disease that is closely related to crystal adhesion and renal tubular epithelial cell impairment. Gap junctions (GJs) and their components (connexins and Cxs) are involved in various pathophysiology processes, but their roles in renal calculi progression are not well defined. Our previous RNA microarray analysis suggests that GJs are one of the key predicted pathways involved in the renal calcium oxalate (CaOx) crystal rat model. In the current study, we found that the Cx43 and Cx32 expression and the GJ function decreased significantly after stimulation with CaOx or sodium oxalate (NaOx) in NRK-52E, MDCK, and HK-2 cells, and Cx43 expression also decreased in renal tissues in renal CaOx crystal model rats. Inhibition of Cx43 in NRK-52E cells by small interference RNA significantly increased the CD44 and androgen receptor expression, and the adhesion between CaOx crystals and cells, which were consistent with the function of GJ inhibitors. On the other hand, after GJ function and Cx43 expression were increased by allicin, diallyl disulfide, or diallyl trisulfide, the impairment of NRK-52E cells by NaOx or other GJ inhibitors and the adhesion between CaOx crystals and renal cells decreased significantly. Furthermore, allicin also increased Cx43 expression and inhibited crystal deposition in rat kidneys. Taken together, our results provide a basis that GJs and Cx43 may participate in renal CaOx stone progression and that allicin, together with its analogues, could be potential drugs for renal calculus precaution.

Gastrin-releasing peptide receptor gene silencing inhibits the development of the epithelial-mesenchymal transition and formation of a calcium oxalate crystal in renal tubular epithelial cells in mice with kidney stones via the PI3K/Akt signaling pathway.

Between 1% and 15% of people are globally affected by kidney stones, and this disease has become more common since the 1970s. Therefore, this study aims to investigate the effects of gastrin-releasing peptide receptor (GRPR) gene silencing via the PI3K/Akt signaling pathway on the development of the epithelial-mesenchymal transition (EMT) and formation of a calcium oxalate crystal in renal tubular epithelial cells (TECs) of kidney stones. A total of 70 clean and healthy C57BL/6J mice were assigned into the normal ( n = 10) and kidney stones groups ( n = 60). The underlying regulatory mechanisms of GRPR were analyzed in concert with the treatment of shGRPR-1, LY294002, and shGRPR-1 + LY294002 in TECs isolated from mice with kidney stones. A series of experiments were conducted for the measurement of urinary oxalate and urinary calcium, the renal calcium salt deposition, the positive rate of GRPR, the expressions of renal TECs related genes and calcium oxalate regulation related genes, and the growth of calcium crystals induced by cells. After treatment of shGRPR-1 and shGRPR-1 + LY294002, levels of urinary oxalate and urinary calcium in the serum, as well as positive rate of GRPR, became relatively low, levels of E-cadherin enhanced, whereas levels of Akt, PI3K, GRPR, extents of PI3K and Akt phosphorylation, α-SMA, Vimentin and FSP-1, OPN, MCP-1, and CD44 decreased and a number of crystals reduced. Taken together, we conclude that GRPR gene silencing suppresses the development of the EMT and formation of the calcium oxalate crystal in renal TECs of kidney stones through the inactivation of the PI3K/Akt signaling pathway.

Seasonal variation in occurrence of oxalate nephrosis in South Australian koalas (Phascolarctos cinereus)

Many koalas (Phascolarctos cinereus) in the Mount Lofty Ranges, South Australia, are affected by oxalate nephrosis, in which renal calcium oxalate deposition occurs. In other species, suboptimal water intake increases the risk of urinary calcium oxalate crystal formation. Koalas principally rely on eucalypt leaf moisture content to maintain hydration but the Mount Lofty Ranges region has hot, dry summers. This study investigates the association between temperature, rainfall and eucalypt leaf moisture and the occurrence of oxalate nephrosis in this population of koalas. Koalas from the Mount Lofty Ranges population that had died or were euthanased between 2008 and 2016 were necropsied and oxalate nephrosis was determined by histopathology (n=50). Leaf moisture content of Mount Lofty eucalypts was determined seasonally. It was found that increased numbers of koalas with oxalate nephrosis died in the months following high mean maximal temperature and in the months following low rainfall. Eucalypt leaf moisture content was not significantly associated with koala deaths. These findings suggest that hot and dry summer/autumn periods contribute to an increased incidence of koala deaths due to oxalate nephrosis. This is probably due to the effects of evaporative water loss and/or lack of access to supplementary drinking water at this time.

Effect of Klotho protein on renal oxidative stress in rat with calcium oxalate nephrolithiasis

Objective To investigate the protective effect and mechanism of Klotho protein on oxidative stress in renal tubular epithelial cells of experimental rat models of renal calcium oxalate stone. Methods The 30 SD rats, 6-8 weeks old, were randomly divided into 3 groups(10 of each), normal control group(group A), calcium oxalate model group(group B), drug plus calcium oxalate model group(group C). Group A was established with physiological saline by garage each day, group B was established with 1% ethylene glycol in drinking water+ 2% ammonium chloride by garage(2 ml/d), group C was established with Fosinopril 2.5mg+ Valsartan 15mg aqueous solution 2 ml by gavage on the basis of group B(2 ml/d). 4 weeks later, the level of malondialdehyde (MDA), superoxide dismutase(SOD), catalase(CAT) and glutathione peroxidase(GSH) in the kidney homogenate were measured by double antibody sandwich enzyme-linked immunosorbent assay (ELISA), Polymerase chain reaction (RT-PCR) was used to measure expression of Klotho and Nrf2 mRNA, and Western Blot was used to measure the expression of Klotho and Nrf2 protein. Results The level of MDA in group B [(12.43±0.43)μmol/mg] was significantly increased compared to group A[(8.67±0.84)μmol/mg, P 0.05). In group A, B, and C, the levels of SOD were (247.89±2.45), (109.54±4.21), and (189.74±10.47)U/mg, respectively; the levels of GSH were (38.98±4.55), (26.87±3.92), and (31.29±2.54)μmol/mg, respectively; CAT were (138.47±8.74), (119.87±8.45), and(127.46±7.45)U/mg, respectively. The levels of SOD, GSH, CAT in group B were significantly lower than that in group A and C, while those in group B were close to group A (P>0.05). The expression of Klotho and Nrf2 mRNA in group B [(0.208±0.036)and (0.499±0.086)] were significantly lower than group A(1.011±0.174 and 1.023±0.139, P<0.05)and group C(1.123±0.248 and 1.023±0.139, P<0.05). The expression of Klotho and Nrf2 protien were also significantly lower than that in group A and C (P<0.05). Conclusions Valsartan and Fosinopril could prevent the formation of renal CaOx stones by upregulating expression of low level Klotho gene induced by ethylene glycol.This effect may be involved with activation of Keapl-Nrf2-ARE signaling pathway. Key words: Klotho; Nuclear factor-E2 related factor 2(Nrf2); Calcium oxalate stone; Oxidative stress

Experimental study on the inhibitory effect of epigallocatechin gallate on renal calcium oxalate crystal formation

Objective To compare the effects of different dosages of epigallocatechin gallate (EGCG) in calcium oxalate stone rate, to expolre content differences in malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and calcium oxalate crystal deposition. Methods Fifty rats were randomly divided into 5 groups: normal control group, calcium oxalate kidney stone model group and different doses of EGCG intervention group. The control group was permitted to drink purified water freely; model group was given 2 ml stone liquid that was prepared with 1% ethylene glycol and 2% ammonium chloride by the lavage. In the EGCG intervention groups, daily doses of 0.1% EGCG (10, 30, and 50 mg/kg) were given introgastrically on the basis treatment in the model group. Twenty-eight days later, the rats were sacrificed and the right kidney was prepared into homogenate. The thiobarbituric acid (TBA) method was used for the determination of MDA. The xanthine oxidase method was applied to detect SOD activity. CAT activity was tested by ultraviolet spectrophotometry. The DTNB method was used to determine GSH-Px activity. The left kidney tissue was stained by HE method, and crystal deposition was observed by polarizing microscopy. Results In the blank control group, MDA concentration was (2.74±0.47) μmol/L, SOD activity (541.71±10.61) Nu/g, CAT activity (44.49±3.45) U/g and GSH-Px activity (41.61±2.50)×103 U/g·protein. As compared with the blank control group, MDA concentration of calcium oxalate kidney stones in the model group was increased significantly, while the activity of SOD (332.44±12.01) Nu/g, CAT (32.15±1.99) U/g and GSH-Px (21.36±1.58)×103 U/g·protein was significantly decreased (P=0.009). In the low-dose EGCG group, the activity of SOD, CAT and GSH-Px was (517.41±28.37) Nu/g, (34.42±2.55) U/g and (23.94±2.95)×103 U/g·protein respectively, that in the middle-dose EGCG group (545.04±23.84), (35.42±2.61) and (25.10±2.64) respectively, and that in the high-dose EGCG group (550.58±16.55) Nu/g, (35.40±2.39) U/g and (26.67±1.93)×103 U/g·protein respectively. As compared with the model group, SOD, GSH-Px and CAT actively was increased in the EGCG groups (P=0.005). The calcium oxalate crystal deposition in the renal tissue was negatively correlated with the dose of EGCG interference (r=-0.483, P= 0.008). Conclusion There were significant differences in MDA, SOD and GSH-Px contents in calcium oxalate stone rats treated with different doses of EGCG, which indicated that EGCG could reduce the formation of calcium oxalate kidney stones by reducing the oxidative damage. Key words: Calcium oxalate; Epigallocatechin gallate; Oxidative damage

SaRNA-mediated activation of TRPV5 reduces renal calcium oxalate deposition in rat via decreasing urinary calcium excretion.

Hypercalciuria is a main risk factor for kidneystone formation. TRPV5is the gatekeeper protein for mediating calcium transport and reabsorption in the kidney. In the present study, we tested the effect of TRPV5 activation with small activating RNA (saRNA), which could induce gene expression by targeting the promoter of the gene, on ethylene glycol (EG)-induced calcium oxalate (CaOx) crystals formation in rat kidney. Five pairs of RNA activation sequences targeting the promoter of rat TRPV5 were designed and synthesized. The synthesized saRNA with the strongest activating effect was selected, and transcellular calcium transportation was tested by Fura-2 analysis. Subsequently, Sprague-Dawley rats were equally divided into three groups and fed with water, 1% EG for 28days after injecting the negative control saRNA, 1% EG for 28days after injecting the selected TRPV5-saRNA, respectively. The CaOx crystal formation and the 24-h urine components were assessed. In vitro study, saRNA ds-320 could significantly activate the expression of TRPV5 and transcellular calcium transportation. In vivo study, after 28days treatment of EG, rats pre-infected with saRNA ds-320 had lower urinary calcium excretion and renal CaOx crystals formation as compared to that pre-infected with negative control saRNA. Activation of TRVP5 with saRNA ds-320 could inhibit EG-induced calcium oxalate crystals formation via promoting urine calcium reabsorption and decreasing urine calcium excretion in rats.

Effect of autophagy inhibitor chloroquine on the renal calcium oxalate crystals formation in rats

Objective To investigate the effect and potential mechanism of autophagy inhibitor chloroquine on the calcium oxalate crystals formation in rats. Methods From September 2016 to October 2016, Thirty healthy male SD rats were randomly divided into 3 groups: control group, model group and chloroquine intervention group. The method to establish calcium oxalate stone model was drinking water with 1% ethylene and 1% ammonium chloride freely. The rats of chloroquine intervention group were treat with chloroquine (40mg/kg·d) by intraperitoneal injection. Modeling was finished after 28 days. The amounts of renal calcium oxalate crystals were detected by polarizing microscope. For all groups, the amounts of autophagosome were detected by transmission electron microscope. Twenty four hour urine compositions for stone risk factors were detected. The expressions of oxidative stress injury related molecular markers (SOD, MCP-1 and 8-OHdG)and the expressions of autophagy markers (LC3 and P62) were detected by immunohistochemistry. The RNA expressions of SLC26A6 in kidney were detected by Real-time PCR. Results Compared to the model group, the amounts of renal calcium oxalate crystals were significantly reduced in chloroquine intervention group(32.37±5.14 vs. 4.18±0.25, P<0.05). Compared to the control group, the level of autophagy was increased in the model group. Compared to the model group, the level of autophagy was inhibited in the chloroquine intervention group. For control group, model group and chloroquine intervention group, the excretion of urinary oxalate were (3.1±1.5)mmol, (22.5±8.1)mmol, (2.8±1.2)mmol, respectively; the excretion of urinary citrate were (63.4±7.4)mmol, (45.9±9.5)mmol, (15.6±8.2)mmol, respectively. Compared to the control group, the amounts of urinary oxalate were significantly elevated in model group(P<0.05), but citrate were significantly reduced in the chloroquine intervention group(P<0.05). For control group, model group and chloroquine intervention group, the expressions of SOD were 42.24±4.16, 19.21±2.25, 39.08±3.53, respectively; the expressions of MCP-1 were 4.02±0.51, 8.45±0.55, 5.52±0.34, respectively; the expressions of 8-OHdG were 7.16±0.54, 11.21±1.12, 8.67±0.34, respectively; the RNA expressions of SLC26A6 were 0.35±0.07, 1.02±0.17, 0.70±0.06, respectively. Compared to the control group, the expressions of SOD were significantly reduced in the model group, but the expressions of MCP-1, 8-OHdG and SLC26A6 were significantly elevated(P<0.05). Compared to the model group, the expressions of SOD were significantly elevated chloroquine intervention group(P<0.05), but the expressions of MCP-1, 8-OHdG and SLC26A6 were significantly elevated(P<0.05). Conclusions The autophagy inhibitor chloroquine could inhibit the formation of calcium oxalate crystals induced by ethylene in rat kidney via inhibit the renal autophagy level and expressions of the SLC26A6, reducing the renal oxidative stress injury and urinary oxalate excretion. Key words: Chloroquine; SLC26A6, autophagy; Calcium oxalate stone; Oxidative stress injury

Induction of apoptosis and autophagy by calcifying nanoparticles in human bladder cancer cells.

Calcifying nanoparticles have been linked to various types of human disease, but how they contribute to disease processes is unclear. Here, we examined whether and how calcifying nanoparticles isolated from patients with kidney stones are cytotoxic to human bladder cancer cells. Calcifying nanoparticles were isolated from midstream urine of patients with renal calcium oxalate stones and examined by electron microscopy. Human bladder cancer cells (EJ cells) were cultured in the presence of calcifying nanoparticles or nanohydroxyapatites for 12 and 72 h and examined for toxicity using the Cell Counting Kit-8, for autophagy using transmission electron microscopy and confocal microscopy, and for apoptosis using fluorescence microscopy, transmission electron microscopy, and flow cytometry. Changes in protein expression were analyzed by Western blotting. The results showed that the size and shape of the isolated calcifying nanoparticles were as expected. Calcifying nanoparticles were cytotoxic to EJ cells, more so than nanohydroxyapatites, and this was due, at least in part, to the production of intracellular reactive oxygen species. Transmission electron microscopy showed that calcifying nanoparticles were packaged into vesicles and autolysosomes. Calcifying nanoparticles induced greater autophagy and apoptosis than nanohydroxyapatites. Our findings demonstrate that calcifying nanoparticles can trigger bladder cancer cell injury by boosting reactive oxygen species production and stimulating autophagy and apoptosis.

Effect of osteopontin knockdown in the kidneys of hyperoxaluric rats on renal calcium oxalate crystal deposition

Objective To address the effects of osteopontin (OPN) on calcium oxalate renal calculus formation. Methods We silenced the OPN gene in rat kidney in vivo using lentivirus vector-deliverd short hairpin RNA (shRNA) targeting OPN through renal venous transduction. On the day 7 after injection with lentivirus, rats were fed on water containing 0.8% glycol and 1% ammonium chloride for 9 days. At the end of the experiments, the rats were killed and the kidney tissues were removed for subsequent analysis. Results The lentivirus vector could effectively infect renal cortex and medulla tissue. OPN-shRNA transfection resulted in significant reduction in the content of OPN. Crystals recorded as the average total pixels (object area) were 20 716±7 755 and 54 921±3 469 in renal cortex sections in the OPN-shRNA group and negative lentivirus vector group, respectively (P<0.01), as well as 18 618±2 285 and 30 475±1 735 in the medulla sections in both groups, respectively (P<0.01). Conclusion Specific knockdown of OPN gene in kidneys of hyperoxaluric rats leads to a decrease in OPN production and significantly inhabits calcium oxalate crystal deposition. OPN may play a certain role in promoting calcium oxalate renal calculus formation. Key words: Osteopontin; Calcium oxalate; Nephrolithiasis; RNA interference

Alteration in Oxidative/nitrosative imbalance, histochemical expression of osteopontin and antiurolithiatic efficacy of Xanthium strumarium (L.) in ethylene glycol induced urolithiasis.

Xanthium strumarium has traditionally been used in the treatment of urolitiasis especially by the rural people in India, but its antiurolithiatic efficacy was not explored scientifically till now. Therefore, the present study was designed to validate the ethnic practice scientifically, and explore the possible antiurolithiatic effect to rationalize its medicinal use. Urolitiasis was induced in hyperoxaluric rat model by giving 0.75% ethylene glycol (EG) for 28days along with 1% ammonium chloride (AC) for first 14days. Antiurolithiatic effect of aqueous-ethanol extract of Xanthium strumarium bur (xanthium) was evaluated based on urine and serum biochemistry, oxidative/nitrosative stress indices, histopathology, kidney calcium and calcium oxalate content and immunohistochemical expression of matrix glycoprotein, osteopontin (OPN). Administration of EG and AC resulted in hyperoxaluria, crystalluria, hypocalciuria, polyurea, raised serum urea, creatinine, erythrocytic lipid peroxidise and nitric oxide, kidney calcium content as well as crystal deposition in kidney section in lithiatic group rats. However, xanthium treatment significantly restored the impairment in above kidney function test as that of standard treatment, cystone. The up-regulation of OPN was also significantly decreased after xanthium treatment. The present findings demonstrate the curative efficacy of xanthium in ethylene glycol induced urolithiasis, possibly mediated through inhibition of various pathways involved in renal calcium oxalate formation, antioxidant property and down regulation of matrix glycoprotein, OPN. Therefore, future studies may be established to evaluate its efficacy and safety for clinical use.

M1/M2-macrophage phenotypes regulate renal calcium oxalate crystal development.

In our previous report, M2-macrophage (Mφs) deficient mice showed increased renal calcium oxalate (CaOx) crystal formation; however, the role of Mφs-related-cytokines and chemokines that affect kidney stone formation remains unknown. Here, we investigated the role of M1/M2s in crystal development by using in vitro and in vivo approaches. The crystal phagocytic rate of bone marrow-derived M2Mφs was higher than that of bone marrow-derived Mφs and M1Mφs and increased on co-culture with renal tubular cells (RTCs). However, the amount of crystal attachment on RTCs reduced on co-culture with M2Mφs. In six hyperoxaluric C57BL/6J mice, M1Mφ transfusion and induction by LPS and IFN-γ facilitated renal crystal formation, whereas M2Mφ transfusion and induction by IL-4 and IL-13 suppressed renal crystal formation compared with the control. These M2Mφ treatments reduced the expression of crystal-related genes, such as osteopontin and CD44, whereas M1Mφ treatment increased the expression of pro-inflammatory and adhesion-related genes such as IL-6, inducible NOS, TNF-α, C3, and VCAM-1. The expression of M2Mφ-related genes was lower whereas that of M1Mφ-related genes was higher in papillary tissue of CaOx stone formers. Overall, our results suggest that renal crystal development is facilitated by M1Mφs, but suppressed by M2Mφs.