Impact of hypoxia and hypercapnia on calcium oxalate toxicity in renal epithelial and interstitial cells

Although there is an ongoing controversy about the primary site of calcium oxalate stone (CaOx) formation, there is some evidence for extratubular crystallization. However, the mechanisms leading to such interstitial calcifications are not clear. Anatomical studies have demonstrated a close association between the renal vasculature and renal tubules. It has been hypothesized that disorders of the vasculature may contribute to renal stone formation. The exceptional papillary environment with low oxygen and high carbon dioxide is of interest in this context and its impact on CaOx toxicity to renal cells has to be evaluated. LLC-PK1, Madin-Darby canine kidney (MDCK), human umbilical vein endothelial (HUVEC) and fibroblast cell lines were exposed to hypoxia (3% O₂) alone, hypercapnia combined with hypoxia (3% O₂, 18% CO₂) or standard culture conditions (20% O₂) for 72 h. Cell survival rates were determined microscopically after 4 h of incubation with CaOx at final concentrations of 1, 2 and 4 mM. DAPI staining and western blot were used to evaluate the induction of apoptosis. We confirmed that CaOx leads to concentration-dependent effects on the viability of the cell lines. HUVECs were most vulnerable to CaOx among the four cell lines. Incubation under hypoxia alone had no impact on CaOx toxicity to any of the cell lines in terms of survival. However, under combined hypoxic and hypercapnic conditions, all cell lines displayed a significant reduction of cell survival compared to room air incubation. Again, this effect was most pronounced for HUVECs. The induction of apoptosis could not be demonstrated in any experimental setting. Combined hypoxia and hypercapnia clearly aggravate CaOx toxicity to renal cell lines. As we could not demonstrate the induction of apoptosis, this effect may be a result of toxic necrosis. Especially the CaOx effect on interstitial cell lines might be of interest in the chronic ischemic papillary environment. An increased toxicity may lead to recurrent stone formation, and vice versa, diseases of the vasculature, like arteriosclerosis, may further promote stone formation by induction of local ischemia. This issue has to be clarified by further studies.     

Effect of second messenger systems on oxalate uptake in renal epithelial cells

The oxalate transport system along with protein phosphorylation appears to be deranged in stone formers. This study was undertaken to characterize in LLC-PK₁ cells in culture the effect of altering specific intracellular second messenger systems on oxalate uptake. Cellular uptake experiments were performed at 37°C in buffer [265 mM mannitol, 5 mM NaOH, 5 mM KOH, 10 mM Ca-EGTA, 25 mM HEPES/TRIS, pH=7.4 or in Hank's balanced salt solution (HBSS)] containing 200 M labeled oxalate (1-¹⁴C, 0.3 Ci). Cells were preincubated with DAG (final concentration of 100 M), phorbol myristate acetate (10 M), forskolin (50 M), 8-bromo-cyclic AMP (50 M), trifluoroperazine (20 M) and low molecular weight heparin (1 mg/ml) for 10 min in the presence and absence of the anion transport inhibitor DIDS (100 M) and the effect(s) on oxalate uptake at 10, 25 and 45 min incubation were determined. Chemicals (DAG, forskolin, TPA and 8-bromo-cAMP) which stimulate protein kinase A or C activity resulted in an increased uptake of oxalate while inhibitors of these systems (trifluoroperazine and low molecular weight heparin) resulted in decreased oxalate uptake. The results dernonstrate that oxalate uptake in renal tubular cells is modulated by protein kinase C and A dependent mechanisms.     

Apoptosis induced by oxalate in human renal tubular epithelial HK-2 cells

Oxalate is not only considered to be one of the main constituents of urinary stones, but it also has toxic effects on renal tubular epithelial cells, affecting the pathogenesis of nephrolithiasis. We tried to elucidate the effects of oxalate on human renal tubular epithelial cells (HK-2 cells). In addition, we investigated whether the toxic effect of oxalate occurs by apoptosis, and determined the expression of Bcl-2 family and caspase 9 proteins known as apoptosis-related protein. HK-2 cells were incubated with different concentrations of oxalate, and the effect of oxalate on the growth of the cells was assessed by MTT assay. A caspase-3 activity assay and TUNEL assay were performed on HK-2 cells after oxalate exposure in order to evaluate apoptosis. Immunoblot analysis of Bax, Bcl-2, Bcl-xL, and caspase-9 were performed. Oxalate exposure resulted in significant growth inhibition of HK-2 cells as oxalate concentrations increased. The toxic effect of oxalate on HK-2 cells was considered to occur through apoptosis, as suggested by the increase of caspase-3 activity. The percentage of positive nuclei stained using the TUNEL method was 18±2.3 in oxalate-treated cells and 2.5±0.9 in untreated cells (P<0.05). Bax and caspase-9 protein expression increased significantly as oxalate concentrations increased, but Bcl-2 protein expression decreased. There was no difference in Bcl-xL protein expression among the various concentrations of oxalate. Our results show that oxalate has a toxic effect on HK-2 cells and that this effect is induced by apoptosis, which may be mediated by an intrinsic pathway.     

Oxalate transport in cultured porcine renal epithelial cells

Summary Oxalate-containing kidney stones are the most common type (75%) of renal stones. In order to control oxalate excretion in the urine, a basic understanding of the cellular transport of oxalate is imperative. We have utilized the technique of continuous cell culture to establish and characterize a model system to study renal epithelial cell (LLCPK1) oxalate transport. Our data demonstrate that oxalate uptake in these cells is dependent on time, concentration and energy. TheK _m for oxalate uptake was 200 m. Oxalate uptake was decreased at lower temperatures and elevated in an acidic extracellular environment. Both anion exchange inhibitors DIDS and SITS inhibited oxalate oxalate uptake. Sulfate, chloride, and bicarbonate decreased oxalate uptake, as did the diuretics bumetanide and furosemide. There was no evidence for the co-transport of oxalate with sodium. Our data show that monolayers of cultured kidney epithelial cells are a valuable model system for study of the basic cellular mechanisms of oxalate transport.     

正常人和草酸钙结石病人尿草酸钙晶体基质

以改良Ｍｏｒｓｅ和Ｒｅｓｎｉｃｋ法提取１０例上尿路草酸钙结石病人和１１例正常人的尿草酸钙晶体基璺，用双向聚丙烯酰胺凝胶电泳对晶体基质及结晶前后大分子物的蛋白质组成进行了比较分析。     

Madin-Darby canine kidney cells are injured by exposure to oxalate and to calcium oxalate crystals

The reaction of Madin-Darby canine kidney cells (MDCK) to potassium oxalate (KOx), calcium oxalate monohydrate (COM) crystals, or a combination of the two was studied. The most noticeable effect of exposure of the cells to either KOx or COM crystals was loss of cells from the monolayer ranging from 20% to 30%, depending upon the particular treatment. Cellular enzyme values in the media were elevated significantly by 12h of exposure, although in specific instances, elevated levels occurred at earlier time periods. As regards the monolayer, trypan blue exclusion was decreased significantly, although amounting to only a 4–5% reduction. Specific tritiated release occurred at 4 and 12 h after exposure to KOx and at 12 h after exposure to crystals. Structurally, COM-cell interactions were complex and extensive endocytosis was noted. Cells were released from culture either as cellcrystal complexes or from the intercellular spaces after exocytosis. When treatment were combined the effects were only slightly additive, but the two treatments potentiated each other: all media enzyme levels (with one exception) were elevated at 2 h, tritiated adenine release was present at 4 h, and there was more extensive cell loss from the culture monolayer. These data suggest that both KOx and COM crystals damage MDCK cells when applied alone, and in concert they act synergistically.     

金钱草注射液抑制鼠草酸钙结石形成作用的研究

目的　探讨广金钱草防石作用机制。　方法　用金钱草注射液和提取液对草酸钙肾石模型大鼠进行抑制实验研究。　结果　透射电镜显示金钱草能减轻肾小管细胞在乙二醇诱石过程中的崩解、坏死,肾小管腔内一些空泡状膜性囊和致密小体排入减少。注射液组肾组织中草酸和钙含量分别为（１ ．３９７ ±０．８５９）ｍｇ／ｇ 、（３ ．９９３ ±１．２７８）μｍｏｌ／ｇ ,提取液组为（５ ．６６５ ±０ ．６９６） ｍｇ／ｇ、（８．６２１±１ ．４４１）μｍｏｌ／ｇ,均明显低于成石组（ Ｐ＜ ０ ．０５） 。偏光镜观察显示注射液组和提取液组肾中草酸钙晶体形成程度比成石组明显减轻。　结论　金钱草在体内能保护肾组织细胞,对草酸钙晶体形成有明显抑制作用,在防治尿石症方面可能有应用价值     

顺德勒流地区草酸钙结石与饮食危险因素的相关性研究

目的 通过对顺德勒流地区上尿路结石的成分分析,并对草酸钙结石患者进行饮食危险因素的病例对照研究,探讨本地区尿路结石形成的病因学,为临床预防、治疗提供依据。方法 回顾性分析2008 ～2010年156例上尿路结石患者的临床资料,并对结石标本进行化学成分测定对确诊为草酸钙结石患者进行病例对照研究,收集患者饮食危险因素。结果...     

一水草酸钙与二水草酸钙结石形成机理的研究

目的　探讨一水草酸钙（ＣＯＭ） 与二水草酸钙（ＣＯＤ） 结石形成的机制。　方法　应用红外光谱仪对２５８ 块尿结石成分进行检测,同时检测３０ 例患者２４ｈ 尿液生化指标,对测定结果利用ＳＰＳＳ软件进行ｔ 检验。　结果　（１） 尿钙：ＣＯＭ 组（４．８３ ±１ ．９８）ｍ ｍｏｌ／２４ｈ,ＣＯＤ 组（９．８８ ±４ ．２８）ｍｍｏｌ／２４ｈ,Ｐ＜ ０ ．０１ ;（２） 尿磷：ＣＯＭ 组（１９ ．４０ ±９．６９）ｍ ｍｏｌ／２４ｈ,ＣＯＤ 组（２９．２０ ±１２．００）ｍ ｍｏｌ／２４ｈ,Ｐ＜ ０．０５,两组尿钙、尿磷差异有显著性。　结论　二水草酸钙结石患者尿钙、尿磷高于一水草酸钙结石患者,表明二水草酸钙的形成与高钙尿及磷酸盐异质成核有关,而一水草酸钙的形成可能与尿中抑制物缺乏有关。     

通过共培养观察被马兜铃酸活化后的肾小管上皮细胞对肾间质成纤维细胞的作用

目的通过共培养观察被马兜铃酸钠盐(AA-Na)活化后的肾小管上皮细胞株(HK-2)对肾间质成纤维细胞(hRIFs)的作用。方法用AA-Na刺激HK-2,16h后用此活化的HK-2与hRIFs共培养(培养液中加或不加抗TGF-β1抗体),48h后检测hRIFs细胞层中Ⅰ型胶原(ColⅠ)含量。结果(1)AA-Na(20μg/ml)对HK-2无刺激增殖、转分化及细胞毒作用。(2)用此浓度AA-Na刺激HK-216h,能使HK-2分泌TGF-β1显著增加(P<0.05)。(3)用此活化后的HK-2与hRIFs共培养48h,能使后者合成ColⅠ显著增多(P<0.05);而抗TGF-β1抗体(1.0或2.0μg/ml)能部分抑制此反应(P<0.05)。结论被AA-Na刺激活化的HK-2能分泌TGF-β1促进hRIFs合成ColⅠ。     

高草酸尿和输尿管梗阻对鼠肾小管上皮细胞凋亡的影响

目的探讨高草酸尿和输尿管梗阻(UUO)对鼠肾小管上皮细胞凋亡的影响. 方法雄性成年Wistar大鼠40只,随机分为4组,每组10只,A组为对照组,B组为高草酸尿组,C组为输尿管梗阻组,D组为高草酸尿并输尿管梗阻组.所有大鼠均在术后2周处死并取左肾,常规HE染色检测肾小管间质草酸盐结晶沉着情况;原位末端标记法(TUNEL)检测肾小管上皮细胞凋亡情况. 结果 B组和D组组织切片肾小管腔内可见明显草酸盐晶体沉着,A组和C组未见晶体沉着.计数5个高倍镜视野的细胞,A、B、C、D各组肾小管上皮细胞凋亡数分别为1.1±1.1,31.3±8.0,145.0±33.4,275.8±51;A组与各组相比差异均有显著性意义(P<0.001),B组与C组、D组相比差异有显著性意义(P<0.001),C组与D组相比差异亦有显著性意义(P<0.01). 结论高草酸尿可致肾小管间质草酸盐结晶沉着;完全性输尿管梗阻并高草酸尿使肾小管上皮细胞凋亡水平明显增高,且造成肾脏损害,输尿管梗阻是比高草酸尿更重要的促细胞凋亡因素.     

Aspects of the influence of magnesium ions on the formation of calcium oxalate

Summary The influence of magnesium ions on the solubility and formation of calcium oxalate was studied. Both calcium oxalate mineral constituents of urinary calculi (whewellite and weddellite) were prepated in the presence of Mg²⁺-ions. For preparation, a gel growth technique and precipitation in aqueous solutions were used. The metastable weddellite formed only when Mg²⁺ concentration, reaction, temperature and precipitation velocity (see text) were combined in the proper way. It is concluded that Mg²⁺ions may induce an increase of solubility of calcium oxalate but in contrast also broaden the Ostwald-Miers range, thus favouring the formation of larger crystals.     

Effects of luminal oxalate or calcium oxalate on renal tubular cells in culture

Oxalate or calcium oxalate crystal-induced tissue damage could be conducive to renal stone disease. We studied the response of renal proximal (LLC-PK1 and MDCK-II) and collecting (RCCD1 and MDCK-I) tubule cell lines to oxalate ions as well as to calcium oxalate monohydrate (COM) crystals. Cells grown on tissue culture plastic or permeable growth substrates were exposed to high (1 mM) and extremely high (5 and 10 mM) oxalate concentrations, or to a relatively large quantity of crystals (146 µg), after which cell morphology, prostaglandin E₂ (PGE₂) secretion, [³H]thymidine incorporation, total cell numbers and various forms of cell death were studied. Morphological alterations, increased PGE₂ secretion, elevated levels of DNA synthesis and necrotic cell death were induced by extremely high, but not by high oxalate. Crystals were rapidly internalized by proximal tubular cells, which stimulated PGE₂ secretion and DNA synthesis and the release of crystal-containing necrotic cells from the monolayer. Crystals did not bind to, were not taken up by, and did not cause marked responses in collecting tubule cells. These results show that free oxalate is toxic only at supraphysiological concentrations and that calcium oxalate is toxic only to renal tubular cells that usually do not encounter crystals. Based on these results, it is unlikely that oxalate anions or calcium oxalate crystals are responsible for the tissue damage that may precede renal stone formation.     

Effects of oxalate exposure on Madin-Darby canine kidney cells in culture: renal prothrombin fragment-1 mRNA expression

It has been suggested that renal tubular cell damage induced by oxalic acid, one of the components of urinary calculi, may be involved in a variety of ways in the development of urolithiasis. During our study on a calculus related protein, renal prothrombin fragment-1 (RPTF-1), we noted that this is an inflammation related substance that mediates an acute inflammatory reaction, one of the original roles of prothrombin. RPTF-1 is a part of prothrombin that is a coagulation factor known to be expressed in the renal tubule. We examined whether oxalic acid may cause cytotoxic effects on tubular epithelial cells and whether such chemical stimulation may promote the translation of RPTF-1 mRNA into RPTF-1 proteins. We used Madin-Darby canine kidney (MDCK) cells derived from the distal tubule of a dog kidney. In this study, the effects of oxalic acid in culture solution at different concentrations on cytotoxicity were assessed using a MTT assay. The location of active oxygen species was identified using dichlorofluorescein diacetate. After the prothrombin sequence of RPTF-1 was confirmed in MDCK cells, RPTF-1 mRNA expression was determined by RT-PCR. The gene sequence of the same promoter area was ligated, and a luciferase sequence was inserted downstream of the vector. The target sequence was transfected into MDCK cells and the relation between oxalic acid and prothrombin promoter was examined. In addition, the variable expression of RPTF-1 mRNA was quantitatively compared depending on oxalic acid concentrations using real-time PCR. When cytotoxicity was investigated, cells were not damaged but, by contrast, were stimulated and activated under oxalic acid below a certain concentration. The relation between cytotoxicity on the cultured MDCK cell membrane and active oxygen species was confirmed. Luminescence in MDCK cells containing the luciferase gene was detected by the addition of oxalic acid, which activated the prothrombin promoter. A part of the prothrombin gene sequence in the MDCK cells was detected and an increase in the expression of RPTF-1 mRNA in MDCK cells by the addition of oxalic acid was confirmed using real-time PCR. Increased expression of prothrombin by adding oxalic acid has already been demonstrated in previous studies. In this study, however, RPTF-1 mRNA was promoted by oxalic acid and a direct association between oxalic acid and RPTF-1 is indicated. This finding shows that increased oxalic acid in urine induces the expression of RPTF-1 in tubular epithelial cells and thereby causes the generation of active oxygen species.     

复发性草酸钙结石与尿内酸性粘多糖的关系

为了探讨酸性粘多糖（ＧＡＧｓ）对草酸钙结石形成的抑制作用机理，收集了１２例正常人及１５例复发性草酸钙结石病人２４小时尿，经超滤后，用ＰｒｏｎａｓｅＥ蛋白酶降解尿中糖蛋白，纯化ＧＡＧｓ，并分别测定了蛋白水解前后尿样中ＧＡＧｓ含量及种晶体系下ＧＡＧｓ对草酸钙晶体粒度分布。结果表明：（１）水解前后，正常人尿中ＧＡＧｓ含量均比结石病人高；（２）草酸钙晶体生长抑制指数（Ｉｇ）、聚集抑制指数（Ｉａ）与ＧＡＧｓ的含量成正比。     

Dietary oxalate loads and renal oxalate handling

PURPOSE: Dietary oxalate makes a significant contribution to urinary oxalate excretion and, thus, may have a role in calcium oxalate kidney stone formation. Studies have indicated that the ingestion of oxalate rich foods results in transient increases in plasma oxalate concentrations and urinary oxalate excretion. We examined changes in plasma and urinary oxalate following oral crystalline oxalate loading under controlled dietary conditions to further define the renal handling of oxalate by normal adults. MATERIALS AND METHODS: Six normal adult subjects consumed controlled diets of known oxalate content for 1 week before ingesting loads of 0, 2, 4 and 8 mmol of oxalate. Urinary and plasma changes were measured to assess renal oxalate handling. Urinary excretion of proximal tubule derived enzymes and isoprostanes was monitored to assess for renal injury and oxidative stress. RESULTS: Time and dose dependent changes in plasma oxalate, urinary oxalate and in the clearance ratio of oxalate-to-creatinine were observed. A significant correlation (r=0.43, p <0.001) between the oxalate-to-creatinine clearance ratio and plasma oxalate levels was identified. No changes in urinary markers of oxidative stress or renal injury were observed following the 8 mmol oxalate load. CONCLUSIONS: Oxalate is rapidly absorbed and cleared by the kidney by filtration and secretion following an oral oxalate load. Renal oxalate secretion has a significant role in the renal handling of an oral oxalate load. There is no evidence of acute renal injury or oxidative stress with oral oxalate loads in these experimental conditions.     

The impact of dietary oxalate on kidney stone formation

The role of dietary oxalate in calcium oxalate kidney stone formation remains unclear. However, due to the risk for stone disease that is associated with a low calcium intake, dietary oxalate is believed to be an important contributing factor. In this review, we have examined the available evidence related to the ingestion of dietary oxalate, its intestinal absorption, and its handling by the kidney. The only difference identified to date between normal individuals and those who form stones is in the intestinal absorption of oxalate. Differences in dietary oxalate intake and in renal oxalate excretion are two other parameters that are likely to receive close scrutiny in the near future, because the research tools required for these investigations are now available. Such research, together with more extensive examinations of intestinal oxalate absorption, should help clarify the role of dietary oxalate in stone formation.     

Fibronectin inhibits endocytosis of calcium oxalate crystals by renal tubular cells

BACKGROUND: Fibronectin (FN; 230 kDa) is a multifunctional alpha2-glycoprotein distributed throughout the extracellular matrix and body fluids. We recently reported that FN has a protective effect against injury of renal tubular cells by exposure to oxalate and calcium oxalate (CaOX) crystals and inhibits the adhesion of CaOX crystals to renal tubular cells. In the study presented here, we investigated whether FN has inhibitory effect on crystal endocytosis by renal tubular cells. METHODS: The inhibitory effect of FN on endocytosis of CaOX crystals by MDCK cells was examined by using a radioactivity uptake assay. Also, crystal endocytosis by cells was morphologically assessed by means of transmission electron microscopy (TEM). RESULTS: FN had inhibitory effects on CaOX crystal endocytosis by MDCK cells. The morphological TEM study showed that few crystals were taken into cells when FN was added compared to the number of crystals when FN was not added. CONCLUSION: We found that FN had the inhibitory effects on the interaction between crystals and renal tubular cells, including the adhesion or endocytosis of crystals by cells.     

鱼油抑制实验鼠草酸钙结晶形成

目的 了解鱼油在尿石形成中的作用。方法 ６０只大鼠随机分４组,饮用１％乙二醇（ＥＧ）水,同时喂饲不同剂量的鱼油。４周后检测各组大鼠肾功能、草酸钙结晶、２４小时尿钙和尿草酸。结果 加服鱼油组鼠肾积水、组织水肿减轻,肾组织内草酸钙结晶数及含钙量明显减少,２４小时尿钙排出减少;尿尿素氮、肌酐排出明显增加,而血中尿素氮、肌酐浓度显著低于成石组。结论 鱼油能抑制实验性高草酸尿症大鼠体内草酸钙结晶形成,减少尿