Oxalate regulates crystal-cell adhesion and macrophage metabolism via JPT2/PI3K/AKT signaling to promote the progression of kidney stones

Oxalate is an organic dicarboxylic acid that is a common component of plant foods.The kidneys are essential organs for oxalate excretion,but excessive oxalates may induce kidney stones.Jupiter microtubule associated homolog 2(JPT2)is a critical molecule in Ca^(2+)mobilization,and its intrinsic mechanism in oxalate exposure and kidney stones remains unclear.This study aimed to reveal the mechanism of JPT2 in oxalate exposure and kidney stones.Genetic approaches were used to control JPT2 expression in cells and mice,and the JPT2 mechanism of action was analyzed using transcriptomics and untargeted metabolomics.The results showed that oxalate exposure triggered the upregulation of JPT2,which is involved in nicotinic acid adenine dinucleotide phosphate(NAADP)-mediated Ca^(2+)mobilization.Transcriptomic analysis revealed that cell adhesion and macrophage inflammatory polarization were inhibited by JPT2 knockdown,and these were dominated by phosphatidylinositol 3-kinase(PI3K)/AKT signaling,respectively.Untargeted metabolomics indicated that JPT2 knockdown inhibited the production of succinic acid semialdehyde(SSA)in macrophages.Furthermore,JPT2 deficiency in mice inhibited kidney stones mineralization.In conclusion,this study demonstrates that oxalate exposure facilitates kidney stones by promoting crystal-cell adhesion,and modulating macrophage metabolism and inflammatory polarization via JPT2/PI3K/AKT signaling.

Proposal for pathogenesis-based treatment options to reduce calcium oxalate stone recurrence

Objective:Prevalence of kidney stone disease continues to increase globally with recurrence rates between 30%and 50%despite technological and scientific advances.Reduction in recurrence would improve patient outcomes and reduce cost and stone morbidities.Our objective was to review results of experimental studies performed to determine the efficacy of readily available compounds that can be used to prevent recurrence.Methods: All relevant literature up to October 2020,listed in PubMed is reviewed.Results: Clinical guidelines endorse the use of evidence-based medications,such as alkaline agents and thiazides,to reduce urinary mineral supersaturation and recurrence.However,there may be additional steps during stone pathogenesis where medications could moderate stone risk.Idiopathic calcium oxalate stones grow attached to Randall’s plaques or plugs.Results of clinical and experimental studies suggest involvement of reactive oxygen species and oxidative stress in the formation of both the plaques and plugs.The renin-angiotensin-aldosterone system(RAAS),nicotinamide adenine dinucleotide phosphate(NADPH)oxidase,mitochondria,and NOD-like receptor pyrin domain containing-3(NLRP3)inflammasome have all been implicated at specific steps during stone pathogenesis in animal models.Conclusion: In addition to supersaturation-reducing therapies,the use of anti-oxidants,free radical scavengers,and inhibitors of NADPH oxidase,NLRP3 inflammasome,and RAAS may prove beneficial for stone prevention.Compounds such as statins and angiotensin converting enzyme inhibitors are already in use as therapeutics for hypertension and cardio-vascular disease and have previously shown to reduce calcium oxalate nephrolithiasis in rats.Although clinical evidence for their use in stone prevention in humans is limited,experimental data support they be considered along with standard evidence-based medications and clinical expertise when patients are being counselled for stone prevention.

Antilithic Effects of Extracts from Urtica dentata Hand on Calcium Oxalate Urinary Stones in Rats

This study examined the potential antilithic effects of a traditional Chinese medicine Urtica dentata Hand （UDH） in experimental rats and screened the optimal extract of UDH as a possible therapeutic agent for kidney stones. The rat model of urinary calcium oxalate stones was induced by intragastric （i.g.） administration of 2 mL of 1.25% ethylene glycol （EG） and 1% ammonium chloride （AC） for 28 days and was confirmed by Color Doppler ultrasound imaging. The rats in different experimental groups were then intragastrically given petroleum ether extract （PEE）, N-butanol extract （NBE）, aqueous extract （AqE） of UDH, Jieshitong （positive control drug）, and saline, respectively. Treatment with NBE significantly reduced the elevated levels of urinary calcium, uric acid, phosphate, as well as increased urinary output. Accordingly, the increased calcium, oxalate levels and the number of calcium oxalate crystals deposits were remarkably reverted in the renal tissue of NBE-treated rats. In addition, NBE also prevented the impairment of renal function to decrease the contents of blood urea nitrogen （BUN） and creatinine. Taken together, these data suggest that NBE of UDH has a beneficial effect on calcium oxalate urinary stones in rats by flushing the stones out and protecting renal function.

The Deleterious Effect of Inappropriate Calcium and Vitamin D Supplementation during Pregnancy in Women Predisposed to Calcium Oxalate Urolithiasis

Background: Gestational formation of new urolithiasis is rare yet the impact of inappropriate gestational calcium and vitamin D supplementation (Ca/DS) is underestimated. Patients and Methods: we retrospectively evaluated 75 pregnant women with history of UL, yet were stable for >2 years on dietary restrictions, for new UL after Ca/DS. Results: During the past 5 years 21 (48%) of those who had received Ca/DS had developed UL and all had high Vitamin D with hypercalcemia while the remaining 31 patients, without Ca/DS, did not have UL and maintained normal vitamin D urinary calcium without need for supplementation. Overt UL was evident by 30th weeks of gestations and most were diagnosed by ultrasonography and managed by medical expulsive therapy. Conclusion: in patients with history of UL, prudent use of Ca/DS is indicated to avoid new UL.

Involvement of VKORC1 in the Inhibition of Calcium Oxalate Crystal Formation in HK-2 Cells

Summary： The vitamin K epoxide reductase complex subunit 1 （VKORC1）, the rate-limiting enzyme for vitamin K recycling, is significantly down-regulated in the kidneys of urolithiasis patients. This study searched for direct evidence to define the inhibitory activity of VKORC1 against calcium oxalate （CaOx） crystal formation. In the experiment of VKORC1 overexpression, HK-2 cells were transfected with the pFLAG-CMV-7.1-VKORC1 plasmid as a pFLAG-CMV-7.1-VKORC1 transfection group or the pFLAG-CMV-7.1 plasmid as a pFLAG-CMV-7.1 control group. In the experiment of VKORC1 knockdown, HK-2 cells were transfected with the PGPU6/GFP/Neo-VKORClshRNA-2 as a PGPU6/GFP/Neo-VKORClshRNA-2 transfection group or the PGPU6/GFP/Neo-shRNA-NC plasmid as a PGPU6/GFP/Neo-shRNA-NC control group. The expression of VKORC1 in HK-2 cells was detected by real-time quantitative PCR and Western blotting. The CaOx crystal formation was observed under the laser-scanning confocal microscope. It was found that the expression levels of VKORC1 mRNA and protein were significantly higher in the pFLAG-CMV-7.1-VKORC 1 transfection group than in the pFLAG-CMV-7.1 control group （P〈0.01）. The number of CaOx crystals in HK-2 cells incubated in fluorescently labeled CaOx monohydrate （COM） crystal medium for 48 h was 14±4 per field （100×） in the pFLAG-CMV-7.1-VKORC1 transfection group and 26±5 per field （100×） in the pFLAG-CMV-7.1 control group respectively under the laser-scanning confocal microscope. The amount of CaOx crystal aggregation and formation in the pFLAG-CMV-7.1-VKORC 1 transfection group was significantly reduced as compared with the pFLAG-CMV-7.1 control group （P〈0.05）. The expression levels of VKORC 1 mRNA and protein were significantly lower in the PGPU6/GFP/Neo-VKORC 1 shRNA-2 transfection group than in the PGPU6/GFP/Neo-shRNA-NC control group （P〈0.05）. The number of CaOx crystals in HK-2 cells incubated in fluorescently labeled COM crystal medium was 65±11 per field （100x） in the PGPU6/GFP/Neo-VKORC 1 shRNA-2 transfection group and 24±6 per field （100×） in the PGPU6/GFP/Neo-shRNA-NC control group respectively under the laser-scanning confocal microscope. The amount of CaOx crystal aggregation and formation in the PGPU6/GFP/Neo-VKORClshRNA-2 transfection group was significantly increased as compared with the PGPU6/GFP/Neo-shRNA-NC control group （P〈0.05）. These findings suggested that the VKORC 1 protein could inhibit CaOx salt crystallization, adhesion and aggregation. This research would help us to understand the mechanisms involving the interaction between crystallization and epithelial cells and the formation of CaOx. Key words： calcium oxalate crystals; kidney stone; vitamin K epoxide reduetase complex subunit 1; laser-scanning confocal microscopy

Effectiveness of Using Renalof®in the Elimination of Kidney Stones under 10 mm Located in the Renal-Ureteral Tract

Background: In view of the high recurrence of kidney stones in patients, the search for less aggressive, preventive treatments has become increasingly essential. Renalof® offers a phytotherapy alternative. Due to its diuretic and kidney stone demineralisation properties, it has been widely used in this patient population, disintegrating and eliminating calcium oxalate and struvite kidney stones painlessly in the genitourinary system. Methods: A Phase II, randomised, prospective, observational, single-blind study with two treatment arms was conducted in order to determine the efficacy of this alternative therapy: a total of 155 patients were enrolled, 120 were assigned to a Renalof® treatment group and 35 to the placebo group. All were over 18 years of age, of both genders, diagnosed with kidney stones of under 10 mm in diameter, present along the entire renal-ureteral-vesicular tract, diagnosed by ultrasound and renal CAT scan. Divided into two study arms, 120 were administered a dosage of a single 325 mg capsule of the Renalof® product half an hour before the two main meals for 3 months. The presence of kidney stones in any part of the renal-ureteral tract was assessed at monthly consultations using one of the above-mentioned diagnostic tools. Results: Study results show a high rate of effectiveness with Renalof®, finding up to a 65% expulsion rate (78 patients) in the first 8 weeks of treatment, compared to 11.4% (4 patients) in the placebo control group, P < 0.001. It is likely a longer follow-up period would be necessary in patients with kidney stones of 10 mm in diameter or larger than the period applied in the study. Discussion: We strongly recommend the inclusion of this product in kidney stone disease management protocols, especially for patients with kidney stones under 10 mm in diameter, where high response and effectiveness have been observed. Thus, it should be evaluated to reduce surgical treatment costs, as well as those for possible colic episodes and other associated complications.

A Review on Kidney Stone and Its Herbal Treatment

Medicinal plants have been valued for millennia as a rich source of therapeutic compounds for the prevention of various ailments all throughout the world.Kidney stones and urinary calculi affect a huge percentage of the population nowadays.Stone sickness has become more prevalent as a result of changes in living conditions,such as industrialization and hunger.The most common stone recorded in India is calcium oxalate kidney stones.Changes in prevalence and incidence,the occurrence of stone kinds and stone position,and stone removal treatment are all discussed.Medicinal herbs have been utilised for centuries because they are safer,more effective,culturally acceptable,and have less adverse effects than manufactured medications.Patients are advised to consume a low-fat diet,as well as fibres from naturally occurring plants and herbal treatments.The current article discusses the steps that should be taken to maximise the potential of medicinal plants for stone dissolving action.Combining herbal remedies with allopathic treatment is an excellent way to eliminate all issues associated with kidney stones.The purpose of this article is to emphasise the use of herbs as a treatment for urinary stones.

Inhibition of the Crystal Growth and Aggregation of Calcium Oxalate by Algae Sulfated Polysaccharide In-vitro

The influence of sulfated polysaccharide （SPS） isolated from marine algae Sargassum fusiforme on the morphology and phase compositions of urinary crystal calcium oxalate was investigated in vitro by means of scanning electron microscopy and X-ray diffraction. SPS maybe is a potential inhibitor to CaOxa urinary stones by inhibiting the growth of calcium oxalate monohydrate （COM）, preventing the aggregation of COM, and inducing the formation of calcium oxalate dihydrate （COD） crystals.

Impact of the adherence to medical treatment on the main urinary metabolic disorders in patients with kidney stones

Objective:To assess the effect of the adherence to medical treatment on urinary parameters in the 24-h metabolic study of patients with kidney stones.Methods:A retrospective,longitudinal,descriptive,and observational study was carried out by reviewing the hospital electronic medical record from 2014 to 2018.The adherence to drug treatment was measured 6 months after its initiation,and the numerical values of the metabolic studies were compared.Wilcoxon tests were performed to compare the difference before and after treatment.Results:Ninety patients were evaluated,with 73.3% of adherence.The 180-day overall adherence rate was 61.2% in patients treated with a single drug and 85.4% in patients treated with multiple drugs.There is a statistically significant increase in citrate levels in patients with good adherence in comparison with non-adherent patients(p=0.031 vs.p=0.528).Conclusions:Medical treatment and dietary measures in patients with kidney stones have an initial impact at 6 months on the values of the main urinary metabolic alterations that predispose to calculi formation;the most significant is seen in those patients with adherence to medical treatment for hypocitraturia.

The Effect of Potassium Citrate on Simultaneous Growth of Calcium Oxalate Mono-, Di-, and Trihydrate in Synthetic Urine

The inducing effect of potassium citrate （K3cit） on simultaneous growth of calcium oxalate mono-（COM）, di-（COD）, and trihydrate （COT） crystals in synthetic urine was observed with double diffusion gelatinous technique. K3cit can induce the formation of COD and COT, inhibit the aggregation and decrease the surface area of COM crystals. It supported the clinical use of K3cit and may provide important clues to this disease in cure and in search for new drugs.

The Intake of Vitamin C Increases the Risk of Kidney Stone:A review

Vitamin C is an essential vitamin for people,but it possibly linked to an increased risk of kidney stones.This view is controversial.The purpose of this assess was to illuminate the relationship between vitamin C intake and kidney stones.This paper reviewed relevant articles in the past 40 years from pubmed,analyzed the correlation between vitamin C intake and kidney stones,and pointed out the problems in the research process,which is of great reference significance.

Effects of lemon-tomato juice consumption on crystal formation in the urine of patients with calcium oxalate stones:A randomized crossover clinical trial

Background:Dietary supplementation with citrate-containing juices may serve as an effective alternative to potassium citrate therapy for preventing calcium oxalate stone recurrence.This study was performed to evaluate whether consumption of lemon-tomato juice can decrease the tendency for stone formation in the urine of calcium oxalate stone formers.Materials and methods:The study was conducted as a prospective interventional randomized crossover clinical trial with a repeated-measures design.Twenty-two patients with calcium oxalate stones and no metabolic abnormalities in the urine treated with lithotripsy at a tertiary care center from August 2017 to July 2018 were recruited.After a 14-hour overnight fasting,urine samples were collected after the patients consumed either milk only or milk and lemon-tomato juice.Their urine was tested for multiple parameters,including urine pH,specific gravity,calcium-creatinine ratio,and supersaturation with sodium oxalate,followed by optical density measurement via spectrophotometry.Results:There were no significant differences in the background characteristics between the 2 groups.The optical density of the urine samples obtained after consumption of milk only was significantly higher than that after consumption of milk and lemon-tomato juice(mean=0.131 for milk only vs.0.053 for milk and lemon-tomato juice,p<0.001).The urine calcium-creatinine ratio was similar between the groups(mean=0.141 for milk only vs.0.076 for milk and lemon-tomato juice,p=0.019).Conclusions:The addition of lemon-tomato juice as a source of citrate in the diet significantly decreases the established risk factors for calcium oxalate stone formation in patients.This study was prospectively registered at CTRI under number CTRI/2017/04/008312 on April 7,2017.

Simulation of calcium oxalate stone in vitro

Crystallization of calcium oxalate is studied mainly in the diluted healthy urine using scanning electron microscopy (SEM), and is compared with the crystallization in the diluted pathological urine. It suggests that the average sizes of calcium oxalate crystals are not in direct proportion to the concentrations of Ca2+ and Ox2- ions. Only in the concentration range of 0.60-0.90 mmol/L can larger size of CaOx crystals appear. When the concentrations of Ca2+ and Ox2- ions are 1.20, 0.80, 0.60, 0.30 and 0.15 mmol/L in the healthy urine, the average sizes of calcium oxalate crystallites are 9.5 X 6.5, 20.0 X 13.5 and 15.0 jj,m X 10.0 jj,m, respectively, for the former three samples after 6 d crystallization. No crystal appears even after 30 d crystallization for the samples of concentrations of 0.30 and 0.15 mmol/L due to their low supersaturations. The results theoretically explain why the probability of stone forming is clinically not in direct proportion to the concentrations of Ca2+ and Ox2- ions. Laser scattering technology also confirms this point. The reason why healthy human has no risk of urinary stone but stone-formers have is that there are more urinary macromolecules in healthy human urines than that in stone-forming urines. These macromolecules may control the transformation in CaOx crystal structure from monohydrate cal-cium oxalate (COM) to dihydrate calcium oxalate (COD). COD has a weaker affinity for renal tubule cell membranes than COM. No remarkable effect of the crystallization time is observed on the crystal morphology of CaOx. All the crystals are obtuse hexagon. However, the sizes and the number of CaOx crystals can be affected by the crystallization time. In the early stage of crystalli-zation (1-6 d), the sizes of CaOx crystals increase and the number of crystal particles changes little as increasing the crystallization time due to growth control. In the middle and late stages (6-30 d), the number of crystals increases markedly while the growth rate changes little due to the nucleation control.

Effect of Concentration of Structurally-Different Carboxylic Acids on Growth and Aggregation of Calcium Oxalate in Gel Systems

The effect of concentration of structurally-different carboxylic acids such as ethylene diamine tetraacetic acid （H4edta）, citric acid （H3cit）, tartaric acid （H2tart）, and acetic acid （HOAc） on growth and aggregation of calcium oxalate （CaOxa） in gel systems was comparatively investigated. H2tart and H3cit could change the morphology of cal- cium oxalate monohydrate （COM） and induce the formation of calcium oxalate dihydrate （COD）. H4edta could induce the formation of COD at a lower concentration of 0.33 mmol/L and have the strongest ability to inhibit aggregation of COM. HOAc inhibited COM aggregation only at a higher concentration than 500 mmol/L. With increasing the number of carboxylic groups in an acid or increasing the concentration of carboxylic acid, the capacity of this acid to induce COD formation and to inhibit growth and aggregation of COM crystals increased. That is, this capacity followed the order： H4edta〉H3cit〉H2tart〉 〉HOAc. The result in this work suggested that the presence of H3cit and H2tart in urine played a role in the natural defense against stone formation.

低表达ACOT4促进草酸钙对HK2细胞损伤和结晶形成

目的探讨酰基辅酶A硫代酯酶4(acyl-CoA thioeaterase 4,ACOT 4)的表达对草酸钙结石形成的影响。方法以人肾小管上皮细胞HK2细胞为研究对象,使用草酸钙处理HK2细胞,并使用siRNA干扰HK2细胞中ACOT4的表达。qPCR和Western blot法检测HK2细胞中基因的表达水平;CCK-8法检测细胞活率;流式细胞术检测细胞凋亡情况;LDH实验检测细胞损伤情况;晶体黏附实验检测HK2细胞对草酸钙结晶的黏附能力。结果草酸钙可以调节HK2细胞中ACOT4的表达;干扰ACOT4可以显著抑制HK2细胞的增殖能力,并且增强草酸钙对HK2细胞的活性降低、损伤和凋亡的影响;同时,干扰ACOT4可以显著促进HK2细胞对草酸钙晶体的黏附能力。结论敲低ACOT4可以促进草酸钙对HK2细胞的损伤并促进HK2细胞对草酸钙晶体的黏附能力。

二甲双胍通过抑制NF-κB/NLRP3通路改善草酸钙诱导人肾小管上皮细胞损伤研究

目的研究二甲双胍(metformin,Met)通过抑制NF-κBNLRP3通路改善草酸钙(Calcium Oxalate,CaOx)诱导人肾小管上皮细胞(HK-2)损伤。方法分别随机将人肾小管上皮细胞分成对照组、实验组(CaOx+HK-2)、高浓度Met组(CaOx+HK-2+1.2 mM Met)、低浓度Met组(CaOx+HK-2+0.80 mM Met)、通路干预组(CaOx+HK-2+PDTC)五组。各组细胞培养24 h,采用酶联免疫吸附测定(Elisa)法检测细胞上清液中炎症因子(IL-6、IL-18、IL-1β),采用逆转录-聚合酶链反应(RT-PCR)法检测细胞中NF-κB、NLRP3、骨桥蛋白(OPN)mRNA。结果各组间细胞上清液IL-6、IL-18、IL-1β含量相比差异均有统计学意义(P均<0.05);各组间细胞中NF-κBmRNA表达量、NLRP3mRNA表达量、OPNmRNA表达量相比差异均有统计学意义(P均<0.05)。结论1.20 mmol/L或0.80 mmol/L Met可以通过抑制NF-κB/NLRP3通路调控下游的炎症相关蛋白及黏附蛋白表达,继而改善CaOx诱导人肾小管上皮细胞损伤及减少草酸钙肾结石形成。

不同羧基含量广金钱草多糖对草酸钙晶体生长、聚集及细胞表面黏附的抑制作用

为了探讨羧基化前后广金钱草多糖(DSPs)对草酸钙(CaC_(2)O_(4))晶体成核、生长和聚集的调控作用,采用FTIR、粉末X射线衍射、扫描电镜、ζ电位和热重分析(TGA)等方法,对具有不同羧基(—COOH)含量(质量分数)的DSPs调控形成的CaC_(2)O_(4)晶体进行表征,比较了各DSPs对纳米一水合草酸钙(nano-COM)损伤后HK-2细胞(人肾近端曲小管上皮细胞)表面的黏附分子的表达差异和nano-COM在细胞表面的黏附差异。结果表明,DSPs均可抑制COM晶体生长,并诱导二水合草酸钙(COD)晶体形成,同时增加CaC_(2)O_(4)晶体表面的ζ电位绝对值,抑制晶体间的聚集。随着多糖中—COOH含量从1.17%增加至7.45%、12.2%和17.7%,相应的多糖DSP0、DSP1、DSP2和DSP3对CaC_(2)O_(4)晶体生长的调控能力依次增强。TGA表明晶体中吸附有多糖,在0.2 g·L^(-1)DSPs存在下,掺入晶体中的DSP0、DSP1、DSP2和DSP3的质量分数分别为1.54%、2.94%、7.96%和8.12%。细胞实验表明,DSPs可显著降低HK-2细胞表面黏附分子CD44和Annexin A2的表达,并减少nano-COM在细胞表面的黏附量。DSPs能够抑制CaC_(2)O_(4)晶体成核、聚集、生长及其在肾上皮细胞表面的黏附,这些均有利于抑制CaC_(2)O_(4)肾结石的形成,其中羧基化程度最高的DSP3的能效最佳。

肠道微生物网络在高草酸诱导大鼠肾损伤中的保护作用

目的探讨肠道菌群移植(FMT)对高草酸饮食大鼠草酸代谢的影响及肾脏保护作用。方法将24只雄性SD大鼠随机分为SC组、SC+FMT组、OD+PBS组及OD+FMT组。其中,SC组为对照组,正常大鼠饲料喂养;OD+PBS组及OD+FMT组采用5%草酸含量的饲料喂养。第14天起,OD+PBS组、OD+FMT组及SC+FMT组分别给予PBS溶液及豚鼠粪便滤液灌胃,连续7 d。收集各组大鼠的24 h尿液、粪便及静脉血清,测定肠道菌群及血清尿液生化指标;取大鼠肾脏进行实时定量PCR及免疫组化检测肾素、ACE及OPN的表达情况。结果肠道菌群移植影响了大鼠的肠道菌群,SC+FMT组大鼠的肠道菌群偏离了SC组大鼠的肠道菌群并与豚鼠肠道菌群的相似度提高;与OD+PBS组比较,OD+FMT组的尿草酸、尿素、尿酸、肌酐、血尿素氮/肌酐以及血尿酸明显降低。此外,经FMT治疗后,与OD+PBS组相比,OD+FMT组上调的肾素mRNA表达的被降低,下调的OPN mRNA表达得到了恢复;免疫组化得到了类似的结果。结论肠道菌群移植激活了大鼠肠道中以Muribaculaceae等草酸降解菌为代表的微生物网络。高草酸诱导的肾脏损伤因微生物网络对草酸的降解而恢复,肠道菌群移植对大鼠肾脏具有保护作用。

p38 MAPK通路调控自噬-内质网应激途径介导草酸钙肾结石形成的机制研究

目的 探究p38 MAPK通路对大鼠草酸钙(CaOx)肾结石形成的影响及其作用机制,为肾结石的治疗选择提供新的思路。方法 将40只大鼠分为对照组、SB203580组、CaOx组、SB203580+CaOx组,每组10只,CaOx组和SB203580+CaOx组以1%乙二醇与1%氯化铵配制的混合液灌胃建立CaOx肾结石模型,对照组和SB203580组以饮用水灌胃;造模后,SB203580组和SB203580+CaOx组腹腔注入剂量为5 mg/kg的SB203580,1次/d,连续注射14 d,对照组和CaOx组腹腔注入等体积生理盐水。称量大鼠肾脏质量并计算肾脏系数,全自动生化分析仪检测血清中血清尿素氮(BUN)和血肌酐(SCr)水平,酶联免疫吸附法(ELISA)测定尿液中中性粒细胞明胶酶相关脂质运载蛋白(NGAL)和肾损伤分子-1(KIM-1)水平,钙盐染色(Von Kossa)观察黑色晶体沉积及组织损伤情况,原位末端标记法(TUNEL)观察肾小管细胞凋亡情况,免疫组化染色检测自噬标记物表达,实时荧光定量聚合酶链反应(RT-qPCR)与蛋白质印迹(Western blotting)测定自噬及内质网应激途径相关分子表达。结果 与CaOx组比较,SB203580+CaOx组大鼠造模后体质量较高(P<0.05),肾脏质量、肾脏系数、BUN、SCr、NGAL、KIM-1水平均较低(P<0.05),肾脏组织病理损伤减轻且黑色结晶明显减少,TUNEL阳性细胞比例、LC3B与Beclin-1阳性表达面积占比、LC3B、Beclin-1、CHOP、GRP78 mRNA表达、LC3-Ⅱ/LC3-Ⅰ蛋白比值、Beclin-1、CHOP、GRP78蛋白表达均下调(P<0.05),p62 mRNA和蛋白表达上调(P<0.05)。结论 p38 MAPK通路参与大鼠CaOx肾结石形成,抑制该通路能够减少肾结石形成,该作用可能与其调控自噬-内质网应激有关。

吴茱萸碱通过抑制氧化应激反应对肾草酸钙结石模型大鼠肾功能的影响

目的:观察吴茱萸碱(EVO)通过抑制氧化应激反应对肾草酸钙结石模型大鼠肾功能的影响。方法:从50只SPF级SD雄性大鼠中随机选出10只大鼠作为空白组,其余大鼠通过灌胃1.25%乙二醇+1%氯化铵混合液构建草酸钙结石模型,造模成功后随机分为模型组、L-EVO组、H-EVO组以及阳性组,每组10只。L-EVO组、H-EVO组分别通过腹腔注射40 mg/kg和80 mg/kg的EVO,阳性组灌胃20 mg/kg的葛根素,每天1次,连续处理4周,空白组、模型组给予等量0.9%氯化钠溶液。检测大鼠尿液以及血清生化指标;HE染色检测肾组织病理变化;Pizzolato染色检测草酸钙晶体沉积。结果:组织病理学检查显示,空白组大鼠肾组织染色清晰,均无草酸钙沉积、肾小管变性、坏死或炎症,未见草酸钙晶体沉积。与空白组比较,模型组发现了广泛的肾小管内草酸晶体沉积,严重的肾小管变性、坏死,炎性细胞浸润到肾间质以及肾小管囊肿形成。与模型组比较,L-EVO组、H-EVO组、阳性组肾脏结构明显改善。与空白组比较,模型组草酸钙晶体数量、钙、草酸盐、磷酸盐、尿素氮、尿素、尿酸、丙二醛(MDA)、肿瘤坏死因子-α (TNF-α)、白细胞介素-1β (IL-1β)、草酸钙晶体沉积升高(P<0.05),镁、超氧化物歧化酶(SOD)、谷胱甘肽(GSH)含量降低(P<0.05)。与模型组比较,L-EVO组、H-EVO组、阳性组草酸钙晶体数量、钙、草酸盐、磷酸盐、尿素氮、尿素、尿酸、MDA、TNF-α、IL-1β、草酸钙晶体沉积含量降低(P<0.05),镁、SOD、GSH含量升高(P<0.05)。与L-EVO组比较,H-EVO组草酸钙晶体数量、钙、草酸盐、磷酸盐、尿素氮、尿素、尿酸、MDA、TNF-α、IL-1β、草酸钙晶体沉积含量减少(P<0.05),镁、SOD、GSH含量增加(P<0.05)。结论:EVO可通过抑制氧化应激反应改善肾草酸钙结石模型大鼠肾功能。