Effect of autophagy on oxalate-induced toxicity of human proximal renal tubular epithelial cell. / è‡ªå™¬åœ¨è‰é ¸è¯±å¯¼äººè‚¾å°ç®¡ä¸Šçš®ç»†èƒžæŸä¼¤ä¸­çš„ä½œç”¨.

OBJECTIVES: To investigate the role of autophagy in oxalate-induced toxicity of human proximal renal tubular epithelial cell (HK-2). METHODS: HK-2 cells were exposed to oxalate (1 mmol/L) for 2 h and 3-methyladenine (3-MA) was used to inhibit autophagy. Then Western blotting was used to measure the expression of autophagy-related protein LC3II. Cell viability and cell apoptosis were measured by MTT assay and flow cytometry assay, respectively. RESULTS: Cytoplasmic vacuolization was observed in HK-2 cells after treating with oxalate for 2 h. However, 3-MA showed no effects on the formation of cytoplasmic vacuolization regardless of the dose at 1 or 5 mmol/L. The expression of LC3II protein was significantly increased in the HK-2 cells in the presence of oxalate (0.62±0.03 vs 0.35±0.02, P<0.05). The expression of LC3II protein in HK-2 cells was downregulated by 3-MA at both 1 and 5 mmol/L compared with the blank control (0.17±0.03 vs 0.35±0.02, 0.16±0.03 vs 0.35±0.02, both P<0.05). Oxalate-induced upregulation of LC3II was reversed by 3-MA only at the concentration of 5 mmol/L (0.47±0.04 vs 0.62±0.03, P<0.05) rather than 1 mmol/L (0.61±0.04 vs 0.62±0.03, P>0.05). Oxalate attenuated viability [(77.32±2.69)% vs 100%, P<0.05] and increased the apoptosis [(8.32±1.05)% vs (2.36±0.29)%, P<0.05] in HK-2 cells, and these effects were reversed by 3-MA only at the concentration of 5 mmol/L [(91.91±3.36)% vs (77.32±2.69)%, (3.45±0.21)% vs (8.32±1.05)%, respectively, both P<0.05] rather than 1 mmol/L [(80.48±3.41)% vs (77.32±2.69)%, (7.81±0.47)% vs (8.32±1.05)%, both P>0.05, respectively]. CONCLUSIONS: Autophagy of HK-2 cells is enhanced by oxalate at the concentration of 1 mmol/L. Inhibition of 3-MA-induced autophagy protects HK-2 cells from the oxalate-induced cytotoxicity.

Development of End Stage Renal Disease after Long-Term Ingestion of Chaga Mushroom: Case Report and Review of Literature.

Chaga mushrooms are widely used in folk remedies and in alternative medicine. Contrary to many beneficial effects, its adverse effect is rarely reported. We here report a case of end-stage renal disease after long-term taking Chaga mushroom. A 49-year-old Korean man with end stage renal disease (ESRD) was transferred to our hospital. Review of kidney biopsy finding was consistent with chronic tubulointerstitial nephritis with oxalate crystal deposits and drug history revealed long-term exposure to Chaga mushroom powder due to intractable atopic dermatitis. We suspected the association between Chaga mushroom and oxalate nephropathy, and measured the oxalate content of remained Chaga mushroom. The Chaga mushroom had extremely high oxalate content (14.2/100 g). Estimated daily oxalate intake of our case was 2 times for four years and 5 times for one year higher than that of usual diet. Chaga mushroom is a potential risk factor of chronic kidney disease considering high oxalate content. Nephrologist should consider oxalate nephropathy in ESRD patients exposed to Chaga mushrooms.

Telmisartan inhibits oxalate and calcium oxalate crystal-induced epithelial-mesenchymal transformation via PPAR-γ-AKT/STAT3/p38 MAPK-Snail pathway.

AIMS: Telmisartan (TLM), a highly selective angiotensin II type 1 receptor blocker (ARB) and partial PPAR-γ agonist, has versatile beneficial effects against oxidative stress, apoptosis, inflammatory responses and epithelial-mesenchymal transition (EMT). However, its underlying mechanism of inhibiting oxalate and calcium oxalate (CaOx) crystal-induced EMT by activating the PPAR-γ pathway remains unclear. MAIN METHODS: CCK-8 assays were used to evaluate the effects of TLM on cell viability. In addition, intracellular reactive oxygen species (ROS) levels were measured by the cell-permeable fluorogenic probe 2,7-dichlorofluorescein diacetate (DCFH-DA). Wound-healing and Transwell assays were used to evaluate the migration ability of HK2 cells exposed to oxalate. Moreover, immunofluorescence, immunohistochemistry and western blotting were used to examine the expression of E-cadherin, N-cadherin, vimentin and α-SMA and explore the underlying molecular mechanisms in HK2 cells and a stone-forming rat model. KEY FINDINGS: Our results showed that TLM treatment could protect HK2 cells from oxalate-induced cytotoxicity and oxidative stress injury. Additionally, TLM prevented EMT induction by oxalate and CaOx crystals via the PPAR-γ-AKT/STAT3/p38 MAPK-Snail pathway in vitro and in vivo. However, knockdown of PPAR-γ with small interfering RNA or the PPAR-γ-specific antagonist GW9662 abrogated these protective effects of TLM. SIGNIFICANCE: As a PPAR-γ agonist, TLM can ameliorate oxalate and CaOx crystal-induced EMT by exerting an antioxidant effect through the PPAR-γ-AKT/STAT3/p38 MAPK-Snail signaling pathway. Therefore, TLM can block EMT progression and could be a potential therapeutic agent for preventing and treating calcium oxalate urolithiasis formation and recurrence.

P2X7 Receptor Stimulation Is Not Required for Oxalate Crystal-Induced Kidney Injury.

Oxalate crystal-induced renal inflammation is associated with progressive kidney failure due to activation of the NLRP3/CASP-1 inflammasome. It has been suggested previously that purinergic P2X7 receptor signaling is critical for crystal-induced inflammasome activation and renal injury. Therefore, we investigated the role of the P2X7 receptor in response to crystal-induced cytokine release, inflammation, and kidney failure using in vitro and in vivo models. Dendritic cells and macrophages derived from murine bone marrow and human peripheral blood mononucleated cells stimulated with calcium-oxalate crystals, monosodium urate crystals, or ATP lead to the robust release of interleukin-1beta (IL-1ß). Treatment with the P2X7 inhibitor A740003 or the depletion of ATP by apyrase selectively abrogated ATP-induced, but not oxalate and urate crystal-induced IL-1ß release. In line with this finding, dendritic cells derived from bone marrow (BMDCs) from P2X7-/- mice released reduced amounts of IL-1ß following stimulation with ATP, while oxalate and urate crystal-induced IL-1ß release was unaffected. In sharp contrast, BMDCs from Casp1-/- mice exhibited reduced IL-1ß release following either of the three stimulants. In addition, P2X7-/- mice demonstrated similar degrees of crystal deposition, tubular damage and inflammation when compared with WT mice. In line with these findings, increases in plasma creatinine were no different between WT and P2X7-/- mice. In contrast to previous reports, our results indicate that P2X7 receptor is not required for crystal-induced CKD and it is unlikely to be a suitable therapeutic target for crystal-induced progressive kidney disease.

Oxalate nephropathy following vitamin C intake within intensive care unit
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OBJECTIVE: To report a case of acute oxalate nephropathy related to vitamin C intake within the intensive care unit (ICU). DESIGN: Case report. SETTING: ICU and nephrology department of a French university hospital. PATIENT: A 57-year-old woman with septic shock related to Legionella pneumophila pneumonia complicated by acute respiratory distress syndrome and acute kidney injury who required renal replacement therapy for 75 days. MEASUREMENTS AND MAIN RESULTS: A renal biopsy was performed on day 72 because of persistent anuria and because the patient showed characteristic features of severe acute oxalate nephropathy. The only cause identified was vitamin C intake received during hospitalization within the ICU (~ 30 g over 2.5 months). At month 6 after ICU admission, estimated glomerular filtration rate was 24 mL/min/1.73m2. CONCLUSION: Compelling evidence obtained from in-vitro and animal studies suggest that vitamin C, a circulating antioxidant, may be a valuable adjunctive therapy in critically-ill patients. Data from humans are more conflicting. Oxalate, a well-known metabolite of vitamin C, is excreted by the kidneys and can exert a toxic effect on epithelial cells and causes direct tubular damage, and/or it can crystallize within the tubular lumen. This case highlights an under-recognized secondary adverse event from vitamin C given to critically-ill patients. The use of high-dose vitamin C should be prescribed with caution in this population.
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Molecular analysis of oxalate-induced endoplasmic reticulum stress mediated apoptosis in the pathogenesis of kidney stone disease.

Oxalate, a non-essential end product of metabolism, causes hyperoxaluria and eventually calcium oxalate (CaOx) stone disease. Kidney cells exposed to oxalate stress results in generation of reactive oxygen species (ROS) and progression of stone formation. Perturbations in endoplasmic reticulum (ER) result in accumulation of misfolded proteins and Ca2+ ions homeostasis imbalance and serve as a common pathway for various diseases, including kidney disorders. ER stress induces up-regulation of pro-survival protein glucose-regulated protein 78 (GRP78) and pro-apoptotic signaling protein C/EBP homologous protein (CHOP). Since the association of oxalate toxicity and ER stress on renal cell damage is uncertain, the present study is an attempt to elucidate the interaction of GRP78 with oxalate by computational analysis and study the role of ER stress in oxalate-mediated apoptosis in vitro and in vivo. Molecular docking results showed that GRP78-oxalate/CaOx interaction takes place. Oxalate stress significantly up-regulated expression of ER stress markers GRP78 and CHOP both in vitro and in vivo. Exposure of oxalate increased ROS generation and altered antioxidant enzyme activities. N-Acetyl cysteine treatment significantly ameliorated oxalate-mediated oxidative stress and moderately attenuated ER stress marker expression. The result indicates oxalate toxicity initiated oxidative stress-induced ER stress and also activating ER stress mediated apoptosis directly. In addition, the up-regulation of transforming growth factor ß-1 revealed oxalate may induce kidney fibrosis through ER stress-mediated mechanisms. The present study provide insights into the pathogenic role of oxidative and ER stress by oxalate exposure in the formation of calcium oxalate stone.

MitoTEMPO Prevents Oxalate Induced Injury in NRK-52E Cells via Inhibiting Mitochondrial Dysfunction and Modulating Oxidative Stress.

As one of the major risks for urolithiasis, hyperoxaluria can be caused by genetic defect or dietary intake. And high oxalate induced renal epithelial cells injury is related to oxidative stress and mitochondrial dysfunction. Here, we investigated whether MitoTEMPO, a mitochondria-targeted antioxidant, could protect against oxalate mediated injury in NRK-52E cells via inhibiting mitochondrial dysfunction and modulating oxidative stress. MitoSOX Red was used to determine mitochondrial ROS (mtROS) production. Mitochondrial membrane potential (Δψm) and quantification of ATP synthesis were measured to evaluate mitochondrial function. The protein expression of Nox4, Nox2, and p22 was also detected to explore the effect of oxalate and MitoTEMPO on NADPH oxidase. Our results revealed that pretreatment with MitoTEMPO significantly inhibited oxalate induced lactate dehydrogenase (LDH) and malondialdehyde (MDA) release and decreased oxalate induced mtROS generation. Further, MitoTEMPO pretreatment restored disruption of Δψm and decreased ATP synthesis mediated by oxalate. In addition, MitoTEMPO altered the protein expression of Nox4 and p22 and decreased the protein expression of IL-6 and osteopontin (OPN) induced by oxalate. We concluded that MitoTEMPO may be a new candidate to protect against oxalate induced kidney injury as well as urolithiasis.

Antioxidant Pre-Treatment Reduces the Toxic Effects of Oxalate on Renal Epithelial Cells in a Cell Culture Model of Urolithiasis.

Urolithiasis is characterized by the formation and retention of solid crystals within the urinary tract. Kidney stones are mostly composed of calcium oxalate, which predominantly generates free radicals that are toxic to renal tubular cells. The aim of the study is to explore possible effects of antioxidant pre-treatment on inhibition of oxidative stress. Three cell lines were used as in vitro model of urolithiasis: MDCK I, MDCK II and LLC-PK1. Oxidative stress was induced by exposure of cells to sodium oxalate in concentration of 8 mM. In order to prevent oxidative stress, cells were pre-treated with three different concentrations of l-arginine and vitamin E. Oxidative stress was evaluated by determining the expression of superoxide dismutase (SOD), osteopontin (OPN), and by the concentration of glutathione (GSH). In all three cell lines, pre-treatment of antioxidants increased cell survival. Positive correlation of SOD and OPN expression as well as GSH concentration was observed in all groups of cells. Our results indicate that an antioxidant pre-treatment with l-arginine and vitamin E is able to hamper oxalate-induced oxidative stress in kidney epithelial cells and as such could play a role in prevention of urolithiasis.

Oligomeric proanthocyanidins protect against HK-2 cell injury induced by oxalate and calcium oxalate monohydrate crystals.

The purpose of the study was to test whether the antioxidants oligomeric proanthocyanidins (OPCs) could provide protection against oxalate and calcium oxalate monohydrate crystals (COM) toxicity in HK-2 cells. Four groups were chosen for the study: negative control group, positive control group (COM + oxalate), OPCs group (OPCs + COM + oxalate), Vit E group (Vit E + COM + oxalate). HK-2 cells were exposed for 4, 8, 12 and 24 h. The activity of HK-2 cell was assessed by MTT. Cellular injury was assessed by activity of Na(+)/K(+) ATP enzyme. Peroxidation level was assessed by malondialdehyde (MDA) content in medium and activity of superoxide dismutase (SOD). Morphological changes of HK-2 cell after exposed for 4 and 12 h in each group were observed under Transmission electron microscope (TEM). The effects of OPCs and VitE on oxalate- and COM-exposed cells were tested. After exposed to oxalate and COM crystals, activity of cells, Na(+)/K(+) ATP enzyme and SOD enzyme showed a significant reduction, and MDA content in medium was significantly increased. OPCs group: the addition of OPCs significantly increased activity of cell, SOD and Na(+)/K(+) ATP enzyme while MDA content was significantly decreased compared with the positive control group. VitE group: compared with the positive control group, activity of HK-2 cell, Na(+)/K(+) ATP enzyme was not significantly changed while SOD activity was restored, and MDA content was significantly decreased after the addition of Vit E. Morphological structure of HK-2 cell was extremely changed as observed under TEM after exposure to high level of COM crystals and oxalate. After the addition of OPCs or Vit E, amounts of cells with vacuoles formed in cytoplasms, karyotheca dissolved and nucleolus disappeared were less than in positive control group. The morphological structure changing in OPCs group was slighter than that in Vit E group. OPCs and vitamin E administration may prevent oxalate- and COM-mediated peroxidative injury, restoring intracellular antioxidant enzyme activity. The protection rendered by OPCs was greater than that of vitamin E.

C-phycocyanin confers protection against oxalate-mediated oxidative stress and mitochondrial dysfunctions in MDCK cells.

Oxalate toxicity is mediated through generation of reactive oxygen species (ROS) via a process that is partly dependent on mitochondrial dysfunction. Here, we investigated whether C-phycocyanin (CP) could protect against oxidative stress-mediated intracellular damage triggered by oxalate in MDCK cells. DCFDA, a fluorescence-based probe and hexanoyl-lysine adduct (HEL), an oxidative stress marker were used to investigate the effect of CP on oxalate-induced ROS production and membrane lipid peroxidation (LPO). The role of CP against oxalate-induced oxidative stress was studied by the evaluation of mitochondrial membrane potential by JC1 fluorescein staining, quantification of ATP synthesis and stress-induced MAP kinases (JNK/SAPK and ERK1/2). Our results revealed that oxalate-induced cells show markedly increased ROS levels and HEL protein expression that were significantly decreased following pre-treatment with CP. Further, JC1 staining showed that CP pre-treatment conferred significant protection from mitochondrial membrane permeability and increased ATP production in CP-treated cells than oxalate-alone-treated cells. In addition, CP treated cells significantly decreased the expression of phosphorylated JNK/SAPK and ERK1/2 as compared to oxalate-alone-treated cells. We concluded that CP could be used as a potential free radical-scavenging therapeutic strategy against oxidative stress-associated diseases including urolithiasis.

Genome wide analysis of differentially expressed genes in HK-2 cells, a line of human kidney epithelial cells in response to oxalate.

Nephrolithiasis is a multi-factorial disease which, in the majority of cases, involves the renal deposition of calcium oxalate. Oxalate is a metabolic end product excreted primarily by the kidney. Previous studies have shown that elevated levels of oxalate are detrimental to the renal epithelial cells; however, oxalate renal epithelial cell interactions are not completely understood. In this study, we utilized an unbiased approach of gene expression profiling using Affymetrix HG_U133_plus2 gene chips to understand the global gene expression changes in human renal epithelial cells [HK-2] after exposure to oxalate. We analyzed the expression of 47,000 transcripts and variants, including 38,500 well characterized human genes, in the HK2 cells after 4 hours and 24 hours of oxalate exposure. Gene expression was compared among replicates as per the Affymetrix statistical program. Gene expression among various groups was compared using various analytical tools, and differentially expressed genes were classified according to the Gene Ontology Functional Category. The results from this study show that oxalate exposure induces significant expression changes in many genes. We show for the first time that oxalate exposure induces as well as shuts off genes differentially. We found 750 up-regulated and 2276 down-regulated genes which have not been reported before. Our results also show that renal cells exposed to oxalate results in the regulation of genes that are associated with specific molecular function, biological processes, and other cellular components. In addition we have identified a set of 20 genes that is differentially regulated by oxalate irrespective of duration of exposure and may be useful in monitoring oxalate nephrotoxicity. Taken together our studies profile global gene expression changes and provide a unique insight into oxalate renal cell interactions and oxalate nephrotoxicity.

Selective Rac1 inhibition protects renal tubular epithelial cells from oxalate-induced NADPH oxidase-mediated oxidative cell injury.

Oxalate-induced oxidative cell injury is one of the major mechanisms implicated in calcium oxalate nucleation, aggregation and growth of kidney stones. We previously demonstrated that oxalate-induced NADPH oxidase-derived free radicals play a significant role in renal injury. Since NADPH oxidase activation requires several regulatory proteins, the primary goal of this study was to characterize the role of Rac GTPase in oxalate-induced NADPH oxidase-mediated oxidative injury in renal epithelial cells. Our results show that oxalate significantly increased membrane translocation of Rac1 and NADPH oxidase activity of renal epithelial cells in a time-dependent manner. We found that NSC23766, a selective inhibitor of Rac1, blocked oxalate-induced membrane translocation of Rac1 and NADPH oxidase activity. In the absence of Rac1 inhibitor, oxalate exposure significantly increased hydrogen peroxide formation and LDH release in renal epithelial cells. In contrast, Rac1 inhibitor pretreatment, significantly decreased oxalate-induced hydrogen peroxide production and LDH release. Furthermore, PKC α and δ inhibitor, oxalate exposure did not increase Rac1 protein translocation, suggesting that PKC resides upstream from Rac1 in the pathway that regulates NADPH oxidase. In conclusion, our data demonstrate for the first time that Rac1-dependent activation of NADPH oxidase might be a crucial mechanism responsible for oxalate-induced oxidative renal cell injury. These findings suggest that Rac1 signaling plays a key role in oxalate-induced renal injury, and may serve as a potential therapeutic target to prevent calcium oxalate crystal deposition in stone formers and reduce recurrence.

Osteodistrofia fibrosa em equinos criados em pastagem de Panicum maximum cultivar Aruana: relato de casos / Fibrous osteodystrophy in horses raised on Aruana (Panicum maximum) pasture: case reports

Relata-se a ocorrência de osteodistrofia fibrosa em 38 equinos criados em pastagens de Panicum maximum cultivar Aruana, da raça Mangalarga Marchador, provenientes de uma propriedade localizada no estado do Paraná. No exame clínico geral, observou-se aumento bilateral e simétrico dos ossos da face e, também, aumento de volume na porção distal do rádio. A análise radiológica das áreas afetadas demonstrou redução da densidade óssea, e, na bioquímica sanguínea, 24 (63 por cento) animais apresentaram níveis de fósforo elevados. O exame bromatológico revelou níveis elevados de oxalato na pastagem de Aruana. Conclui-se que a ingestão de Aruana, forragem com altos níveis de oxalato, foi responsável pelo aparecimento de lesões de osteodistrofia fibrosa em equinos.

Fibrous osteodystrophy occurrence is reported in 38 Mangalarga Marchador horses raised on Aruana (Panicum maximum) pasture from a breeding farm in Paraná state. Clinical examination showed bilateral and symmetric increase in bones of face and an increase of volume at the radius distal portion. The radiologic analysis revealed bone density reduction in affected areas. Blood biochemist demonstrated high phosphorus levels in 24 animals (63 percent). Analysis of the pasture was performed and high levels of oxalate were found in the Aruana samples. It was concluded that the ingestion of Aruana, a pasture with high oxalate levels, was responsible for the appearing of fibrous osteodystrophy lesions in horses.

Oxalate-mediated oxidant-antioxidant imbalance in erythrocytes: role of N-acetylcysteine.

The present in-vivo study was to observe the effect of N-acetylcysteine (NAC) on oxalate-induced oxidative stress on rat erythrocytes. A total of 15 Wistar rats were divided into three groups. The control group received normal saline by single intraperitoneal injection. Hyperoxaluria was induced by single intraperitoneal (i.p.) dose of sodium oxalate (70 mg/kg body weight in 0.5 mL saline) to a second group. The third group was administered single i.p. dose of NAC according to 200 mg/kg body weight dissolved in 0.5 mL saline, half an hour after oxalate dose. NAC administration normalized antioxidant enzyme activities (superoxide dismutase and catalase) and reduced malondialdehyde content (indicator of lipid peroxidation) in hyperoxaluric rat's red blood cell (RBC) lysate. NAC administration also resulted in a significant improvement of thiol content in RBC lysate via increasing reduced glutathione content and maintaining its redox status. Oxalate-caused alteration of cholesterol/phospholipid ratio (determining membrane fluidity) was also rebalanced by NAC administration. Further, after NAC administration, electron microscopy showed improved cell morphology presenting its prophylactic properties. Above results indicate that NAC treatment is associated with an increase in plasma antioxidant capacity and a reduction in the susceptibility of erythrocyte membranes to oxidation. Thus, the study presents positive pharmacological implications of NAC against oxalate-mediated impairment of erythrocytes.

Synthesis, characterization and in vitro cytotoxicity of the first palladium(II) oxalato complexes involving adenine-based ligands.

The first [Pd(L(n))(2)(ox)] xH(2)O oxalato(ox) complexes involving 2-chloro-N6-(benzyl)-9-isopropyladenine (L(1); complex 1), 2-chloro-N6-(4-methoxybenzyl)-9-isopropyladenine (L(2); 2), 2-chloro-N6-(2,3-dimethoxybenzyl)-9-isopropyladenine (L(3); 3), 2-chloro-N6-(2,4-dimethoxybenzyl)-9-isopropyladenine (L(4); 4), and 2-chloro-N6-(4-methylbenzyl)-9-isopropyladenine (L(5); 5) have been synthesized by the reactions of potassium bis(oxalato)palladate(II) dihydrate, [K(2)Pd(ox)(2)].2H(2)O, with the mentioned organic compounds (H(2)ox=oxalic acid; x=0 for 1-3 and 5 or 2 for 4). Elemental analyses (C, H, N), FTIR, Raman and NMR ((1)H, (13)C, (15)N) spectroscopies, conductivity measurements and thermal studies (thermogravimetric and differential thermal analyses, TG/DTA) have been used to characterize the prepared complexes. The molecular structures of [Pd(L(2))(2)(ox)] (2) and [Pd(L(5))(2)(ox)].L(5).Me(2)CO (5.L(5).Me(2)CO) have been determined by a single crystal X-ray analysis. The geometry of these complexes is slightly distorted square-planar with two appropriate L(n) (n=2 or 5) molecules mutually arranged in the head-to-head (2) or head-to-tail (5) orientation. The L(n) ligands are coordinated to the central Pd(II) ion via the N7 atoms. The same conclusions regarding the binding properties of L(1)-L(5) ligands can be made based on multinuclear NMR spectra. In vitro cytotoxicity of the complexes 1-5 has been evaluated against human chronic myelogenous leukaemia (K562) and human breast adenocarcinoma (MCF7) cancer cell lines. Significant cytotoxicity has been determined for the complexes 3 (IC(50)=6.2 microM) and 5 (IC(50)=6.8 microM) on the MCF7 cell line, which is even better than that found for the well-known and widely-used platinum-bearing antineoplastic drugs, i.e. oxaliplatin and cisplatin.

Oxalate exposure provokes HSP 70 response in LLC-PK1 cells, a line of renal epithelial cells: protective role of HSP 70 against oxalate toxicity.

We investigated the effects of oxalate on immediate early genes (IEGs) and stress protein HSP 70, commonly induced genes in response to a variety of stresses. LLC-PK1 cells were exposed to oxalate. Gene transcription and translation were monitored by Northern and Western blot analysis. RNA and DNA synthesis were assessed by [(3)H]-uridine and [(3)H]-thymidine incorporation, respectively. Oxalate exposure selectively increased the levels of mRNA encoding IEGs c-myc and c-jun as well as stress protein HSP 70. While expression of c-myc and c-jun was rapid (within 15 min to 2 h) and transient, HSP 70 expression was delayed (approximately 8 h) and stable. Furthermore, oxalate exposure resulted in delayed induction of generalized transcription by 18 h and reinitiation of the DNA synthesis by 24 h of oxalate exposure. Moreover, we show that prior induction of HSP 70 by mild hypertonic exposure protected the cells from oxalate toxicity. To the best of our knowledge this is the first study to demonstrate rapid IEG response and delayed heat-shock response to oxalate toxicity and protective role of HSP 70 against oxalate toxicity to renal epithelial cells. Oxalate, a metabolic end product, induces IEGs c-myc and c-jun and a delayed HSP 70 expression; While IEG expression may regulate additional genetic responses to oxalate, increased HSP 70 expression would serve an early protective role during oxalate stress.

Effects of green tea on urinary stone formation: an in vivo and in vitro study.

PURPOSE: We evaluated whether epigallocatechin gallate (EGCG), a main constituent of green tea polyphenols, could protect against cellular toxicity by oxalate and whether green tea supplementation attenuates the development of nephrolithiasis in an animal model. MATERIALS AND METHODS: Cells of the NRK-52E line were incubated with different concentrations of oxalate with and without EGCG, and toxicity and malondialdehyde assays were done to investigate the cytotoxic effect of oxalate and the anti-oxalate effect of EGCG.. In a second series of experiments, male Sprague-Dawley rats were divided into three groups. Group 1 animals (controls) were fed regular chow and drank water ad libitum; group 2 animals were fed chow containing 3% sodium oxalate with the administration of gentamicin (40 mg/kg) and drank water ad libitum; group 3 animals were fed the same diet as group 2 with gentamicin administration and drank only green tea. Rats were killed 4 weeks later after a 24-hour urine collection, and the kidneys were removed for morphologic examination. RESULTS: As oxalate concentrations increased, the number of surviving cells decreased, and the formation of free radicals increased. The administration of EGCG inhibited free-radical production induced by oxalate. Green tea supplementation decreased the excretion of urinary oxalate and the activities of urinary gammaglutamyltranspeptidase and N-acetylglucosaminidase. The number of crystals within kidneys in group 3 was significantly lower than in group 2. CONCLUSIONS: Green tea has an inhibitory effect on urinary stone formation, and the antioxidative action of EGCG is considered to be involved.

Oxalate toxicity in renal cells.

Exposure to oxalate, a constituent of the most common form of kidney stones, generates toxic responses in renal epithelial cells, including altered membrane surface properties and cellular lipids, changes in gene expression, disruption of mitochondrial function, formation of reactive oxygen species and decreased cell viability. Oxalate exposure activates phospholipase A2 (PLA2), which increases two lipid signaling molecules, arachidonic acid and lysophosphatidylcholine (Lyso-PC). PLA2 inhibition blocks, whereas exogenous Lyso-PC or arachidonic acid reproduce many of the effects of oxalate on mitochondrial function, gene expression and cell viability, suggesting that PLA2 activation plays a role in mediating oxalate toxicity. Oxalate exposure also elicits potentially adaptive or protective changes that increase expression of proteins that may prevent crystal formation or attachment. Additional adaptive responses may facilitate removal and replacement of dead or damaged cells. The presence of different inflammatory cells and molecules in the kidneys of rats with hyperoxaluria and in stone patients suggests that inflammatory responses play roles in stone disease. Renal epithelial cells can synthesize a variety of cytokines, chemoattractants and other molecules with the potential to interface with inflammatory cells; moreover, oxalate exposure increases the synthesis of these molecules. The present studies demonstrate that oxalate exposure upregulates cyclooxygenase-2, which catalyzes the rate-limiting step in the synthesis of prostanoids, compounds derived from arachidonic acid that can modify crystal binding and may also influence inflammation. In addition, renal cell oxalate exposure promotes rapid degradation of IkappaBalpha, an endogenous inhibitor of the NF-kappaB transcription factor. A similar response is observed following renal cell exposure to lipopolysaccharide (LPS), a bacterial cell wall component that activates toll-like receptor 4 (TLR4). While TLRs are primarily associated with immune cells, they are also found on many other cell types, including renal epithelial cells, suggesting that TLR signaling could directly impact renal function. Prior exposure of renal epithelial cells to oxalate in vitro produces endotoxin tolerance, i.e. a loss of responsiveness to LPS and conversely, prior exposure to LPS elicits a similar heterologous desensitization to oxalate. Renal cell desensitization to oxalate stimulation may have profound effects on the outcome of renal stone disease by impairing protective responses.