Inhibition Of TMAO Production Attenuates Chronic Kidney Disease And Related Cardiovascular Damage

BackgroundThe relationship between intestinal microflora and human health has attracted extensive attention in recent years.Trimethylamine N-oxide(TMAO)is a metabolite of intestinal microflora.Bacterial metabolism of dietary choline and L-carnitine in the intestine leads to an intermediate,trimethylamine(TMA),which is absorbed from the gut and subsequently oxidized by one or more.hepatic flavin-containing monooxygenases(FMO)to generate TMAO.Several studies have replicated the association of TMAO with cardiovascular disease(CVD).In humans,plasma TMAO levels are elevated in CVD patients compared to controls,exhibit a dose-dependent relationship with severity of atherosclerotic disease,and are associated with increased risk for major adverse cardiovascular events(MACE),such as death,myocardial infarction and stroke.Feeding mice with choline or TMAO leads to significantly increased plasma TMAO levels and atherosclerosis,and induces cardiomyocyte hypertrophy,myocardial fibrosis and poor cardiac function.In the process of TMAO production,the conversion of precursor substances such as choline into TMA by the intestinal flora TMA lyase is the most critical step.Iodomethylcholine(IMC)is a TMA lyase inhibitor of intestinal flora developed by Stanley Hazen et al.through long-term research and design,which could reduce the plasma TMA and TMAO levels and decrease atherosclerosis and thrombosis potential in mice.Chronic kidney disease(CKD)is a serious public health problem that affects all human beings in the world.CKD is closely related to CVD.Researches show that the incidence rate of cardiovascular events increases significantly with the deterioration of renal function in CKD patients.CKD is also associated with premature death with CVD as the leading cause.TMAO in circulation is mainly excreted through the kidney.The decrease of renal function during CKD leads to the decrease of TMAO excretion and insufficient clearance,which leads to the accumulation of TMAO in the body.And high circulating TMAO is associated with an increased risk of CVD and mortality in people with CKD.If TMAO level can be effectively reduced in CKD patients,it is very likely to reduce CKD related cardiovascular damage to a certain extent.On the other hand,feeding mice with choline or TMAO leads to significantly increased renal fibrosis and dysfunction,suggesting that TMAO is not merely a marker but also a cause for CKD,resulting in a vicious circle.Therefore,reducing the level of TMAO in CKD patients may not only improve the cardiovascular complications related to CKD,but also help to protect the kidney itself and delay the progression of CKD.In view of the significant impact of TMAO on human health and disease,researchers have been exploring and searching for the specific molecular mechanism that how TMAO exerts its biological effects.Marcus Seldin et al.have demonstrated that TMAO can promote inflammatory responses in vascular endothelial cells through the activation of NF-?B signaling pathway.Recently,Sifan Chen et al.demonstrated that TMAO can participate in the unfolding protein response(UPR)of the endoplasmic reticulum in hepatocytes,which is,TMAO directly binds to the PERK protein in endoplasmic reticulum and activates PERK signal pathway,causing changes of the related downstream signal transduction pathways.That study provides a new idea for studying the biological effect and molecular mechanism of TMAO.Previous studies have shown that the PERK signaling pathway plays an important role in the activation ofNF-?B:NF-?B is bound by I?B? under normal circumstances to maintain its inactive state.PERK can reduce the transcription level of I?B? by activating its downstream eIF2?,resulting in an increase in the content of unbound free-form NF-?B.So more NF-?B enters the nucleus and get activated.In view of the above theoretical basis,we can't help but think that if TMAO can activate NF-?B signaling pathway,it is likely to be achieved by PERK activation.To this end,from the overall animal level and the cellular level,this project intends to investigate whether the intestinal flora TMA lyase inhibitor IMC can effectively reduce the circulating level of TMAO in CKD mice and whether the inhibition of TMAO can delay CKD progression in mice and improve CKD-related cardiovascular damage.At the same time,in vitro experiments are used to explore the role of PERK and NF-?B signaling pathways in TMAO-induced kidney damage,and to clarify the specific molecular mechanism of TMAO.This study will provide new ideas for revealing the biological effects of TMAO,provide new targets for intervention and treatment of CKD and related cardiovascular damage,and provide a theoretical basis for the clinical application of TMA lyase inhibitors.Objective(1)To observe the relationship between TMAO level and renal injury in an adenine-induced CKD mouse model,and to explore whether IMC can inhibit TMAO production,delay the progression of CKD and improve renal function;(2)To explore the injury effect of TMAO on renal epithelial cells in vitro,observe the changes of PERK and NF-?B signaling pathway and test the blocking effect of related inhibitors;(3)To observe the manifestation and severity of cardiovascular damage in CKD mice,and to determine whether IMC can improve the cardiovascular complications related to CKD by inhibiting TMAO production.Methods1 Mouse modelAnimal experiment 1:Establish CKD mouse model by feeding the mice with adenine diet.Four-week-old healthy female apoE knockout mice were fed the following diets for 14 weeks before tissue collection:(1)chow diet(Con),(2)chow diet+0.06%IMC(Con+IMC),(3)chow diet+0.2%adenine(Ade),and(4)chow diet+0.2%adenine+0.06%IMC(Ade+IMC).At 12th week of diet feeding,mice were individually placed in metabolic cages for collection of urine for 24 hours.At the end of diet feeding period(14 weeks),mice were fasted for 4 hours before blood and tissue collection.Animal experiment 2:Four-week-old healthy female apoE knockout mice were selected and randomly divided into 2 groups according to the following diet:(1)chow diet(Con);(2)chow diet+adenine(Ade).After 24 weeks of feeding,the mice were sacrificed after echocardiography,and blood samples and tissues were collected for subsequent experiments.The adenine content in the diet of Ade group was 0.2%for the first 14 weeks,and was 0.15%for the last 10 weeks.The diet in the Con group remained unchanged.2 EchocardiographyTwo-dimensional and M-mode echocardiography technology was used to observe the left ventricular hypertrophy in mice.The interventricular septum(IVS)thickness,left ventricular internal dimension(LVID),left ventricular posterior wall(LVPW)thickness,left ventricular volume(LV Vol),left ventricular mass(LV mass),left ventricular mass/body weight(LV mass/Body weight),left ventricular ejection fraction(LVEF)and other indicators were measured.3 Biochemical assaysPlasma TMA,TMAO,creatinine,and indoxyl sulfate levels were determined by mass spectrometry.Plasma urea levels were determined using a colorimetric assay.Plasma fibroblast growth factor 23(FGF23)and cystatin C levels were determined by ELISA.Urine albumin and creatinine levels were determined using an ELISA kit and a colorimetric assay kit,respectively.Urine albumin to creatinine ratios(ACR)were then calculated.Total cholesterol(TC),high density lipoprotein cholesterol(HDL-C)and triglyceride(TG)levels were determined by enzymatic colorimetry.The levels of very low density/Intermediate density/Low density lipoprotein cholesterol(VLDL/IDL/LDL-C)were then calculated.TNF-? and IL6 were determined by ELISA method.4 HistologyHistological sections of mice kidneys were stained with Masson's trichrome and quantitated for cortical scar area and collagen deposition in a blinded fashion.Frozen sections of the proximal aorta were stained with Oil Red O and the atherosclerotic lesions were quantitated as the stained area.Frozen sections of the proximal aorta were stained with Alizarin Red S and the calcification was quantitated as the stained area.5 Cell culture and experimentsMurine inner renal medullary collecting duct cells(mIMCD3),normal human primary renal proximal tubule epithelial cells(RPTEC)and Madin-Darby canine kidney ? cells(MDCK ?)were cultured separately.Different doses of TMAO were used to treat the above three types of renal epithelial cells,and the cells were retained for RNA or total protein extraction.Moreover,after cultured in 6-well plates for 24 hours,MDCK ? cells were then divided into four groups:(1)Control group:normal culture medium;(2)Inh group:normal culture medium+100nMNF-?B inhibitor ?;(3)TMAO group:normal culture medium+200?M TMAO;(4)TMAO+Inh group:normal culture medium+2 00?M TMAO+100nM NF-?B inhibitor ?.After 48 hours of stimulation,the cells were retained for RNA extraction.Finally,the mouse RAW264.7 macrophages were cultured and stimulated with different doses of TMAO.The cells were then retained for RNA extraction.6 RNA extraction and quantitative RT-PCRTotal RNA was extracted from tissue or cell samples using the miRNeasy Mini Kit or Qiazol according to the protocols provided by the manufacturers.The cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit.Quantitative RT-PCR was performed using gene-specific primers and the SYBR green master mix in a Roche Lightcycler 480 system.7 RNA sequencingRNA libraries were prepared using the Illumina TruSeq kits.Following barcoding,24 samples per lane were sequenced on a HiSeq4000 using 50bp single-end protocol.Reads were QC'd using FastQC in batch mode and mapped to the mouse genome using STAR aligner version 2.3.1.The count data were normalized using DESeq2's median of ratios method.Differential expression analysis was performed using DEseq2 with statistically significant genes called using adjusted P-value cutoffs of less than 0.1.Gene ontology analysis was performed using DAVID 6.8.The Benjamini-Hochberg method was used to obtain false discovery rates for enriched pathways.8 Western blotWestern blot was performed using proteins from cell lysate described above.Primary antibodies against phospho-NF-?B p65,NF-?B p65,phospho-PERK,PERK,phospho-eIF2?,eIF2?,phospho-IRE1?,IRE1?,ATF4 and GAPDH were used in the experiments.9 Statistical analysesTwo-tailed Student's t-test was utilized to compare means between 2 groups.For experiments with a one-factorial design,one-way ANOVA with Tukey's multiple comparisons test was performed.For experiments with a two-factorial design,two-way ANOVA with Holm-Sidak post hoc analysis was performed.Statistical analyses were performed in GraphPad Prism version 8 and P<0.05 was considered statistically significant.Results1 IMC reduced plasma TMAO levels in CKD micePlasma TMAO levels of the Con+IMC group were significantly lower as compared to the Con group,validating the effectiveness of IMC on inhibition of TMAO production.Plasma TMAO levels of the Ade group were much higher than the Con group,confirming that CKD is associated with elevation of TMAO level.IMC dramatically decreased TMAO levels in Ade+IMC group as compared to the Ade group.There was also a trend of a decrease in TMA levels in the Ade+IMC group as compared to the Ade group.2 Inhibition of TMAO production improved kidney function in CKD miceCirculating levels of markers of kidney disease,including creatinine,urea and cystatin C were significantly elevated in the Ade group as compared to the Con group,whereas those of the Ade+IMC group were significantly decreased as compared to the Ade group,suggesting that IMC attenuated CKD induced by adenine feeding.Circulating levels of a uremic toxin,indoxyl sulfate,were increased in the Ade group as compared to the Con group,as well as the urine ACR,indicating impaired kidney function.Ade+IMC group exhibited significantly decreased indoxyl sulfate and urine ACR as compared to the Ade group,demonstrating significantly improved kidney function in the former.At the same time,there was no significant difference in the average daily food intake of each group of mice,which ruled out the influence of unbalanced dietary intake on the experimental results.3 Inhibition of TMAO production alleviated kidney pathology in CKD miceHistological sections of kidney were stained with Masson's trichrome and quantitated for cortical scar area and collagen deposition.Kidney sections of both Con and Con+IMC groups appeared normal,whereas those of the Ade and Ade+IMC groups showed cortical scar and collagen deposition.Quantification of the kidney pathological changes revealed significant decreases in cortical scar and collagen deposition in the Ade+IMC group as compared to the Ade group.These data demonstrated that IMC decreased renal pathological changes associated with adenine feeding.4 Inhibition of TMAO production reduced kidney inflammation and fibrosis and improved kidney metabolic stateQuantitative RT-PCR analysis revealed that adenine feeding significantly induced the mRNA levels of inflammation genes,Ccl2,Ccl20,and Lcn2,and fibrosis genes,Collal and Col3al,in the kidneys as compared to the Con group.IMC supplementation significantly decreased renal expression of these genes.As compared to Ade group,RNA sequencing and David pathway analysis revealed that Ade+IMC group kidneys had down-regulated clusters in the enrichment of extracellular matrix,protease inhibitor,complement and coagulation cascade,immunity,and cytokine.The up-regulated functional clusters include:lipid metabolism,mitochondria,ion transport,endoplasmic reticulum,and cellular water homeostasis,indicating an improved metabolic state associated with IMC supplementation.5 TMAO induced inflammation in renal epithelial cellsDifferent types of renal epithelial cells were treated with culture media containing different concentrations of TMAO.In mIMCD3 cells,compared with the blank control group,the expression level of Il1? gene in the 20mM TMAO group was significantly up-regulated,and the expression levels of 111?,MIP2 and Lcn2 genes in the 40mM TMAO group were significantly up-regulated.In RPTEC cells,compared with the blank control group,the expression level of CCL2 gene in the 200?M TMAO group was significantly up-regulated,and the expression levels of CCL2 and TNF-? genes in the 400?M TMAO group were both significantly up-regulated.In MDCK II cells,we observed that 50?M,100?M,200?M of TMAO significantly increased the mRNA levels of inflammation genes including IL8,CCL2,and CCL20 as compared to the control group,whereas the mRNA levels of genes involved in UPR,ATF4 and CHOP,were similar between the TMAO treated and control cells.6 TMAO activated NF-?B pathway in renal epithelial cellsMDCKII cells were stimulated with 200?M TMAO for 48 hrs.Western blot was used to detect the protein levels of related pathways.It revealed that NF-?B pathway was activated by TMAO treatment as evidenced by significant increases in phosphorylation of the NF-?B p65 subunit and the p-NF-?B p65 to total NF-?B p65 ratio.In contrast,TMAO treatment did not activate proteins in the UPR pathways,including PERK,eIF2?,IREla and ATF4 in MDCK ? cells.7 NF-?B inhibitor abolished the pro-inflammatory effect of TMAOMDCK ? cells were divided into Control group,Inh group,TMAO group and TMAO+Inh group as described above.Quantitative RT-PCR was used to detect the expression of inflammation genes in all groups.The results showed that the expression of inflammation genes in TMAO group was significantly higher than that of the Control group,while the expression of IL8,CCL2,and CCL20 in TMAO+Inh group was significantly lower than that in TMAO group.These results indicate that NF-?B inhibitor completely abolished the stimulatory effect of TMAO,suggesting the importance of NF-? B in mediating the inflammatory effect of TMAO.8 TMAO also had a pro-inflammatory effect on macrophagesMacrophages play an important role in the development of CKD induced by adenine.In order to clarify whether TMAO also affects macrophages,we incubated RAW264.7 macrophages with 200?M TMAO medium.After 24 hours,we found that the expression levels of inflammation genes in the cells were significantly increased,including Cox2,Il1?,MIP2 and TNF-?.Therefore,TMAO not only has a pro-inflammatory effect on the intrinsic epithelial cells of the kidney,but also induces inflammation of macrophages.9 Inhibition of TMAO production attenuated heart hypertrophy in CKD miceAt the end of 14th week,the body weight and heart weight of mice in Con group,Con+IMC Group,Ade group and Ade+IMC group were respectively recorded,and the heart/body weight ratio was calculated.Mice fed the adenine diet exhibited significantly increased heart/body weight ratio as compared to those of the Con,Con+IMC,and Ade+IMC groups,whereas no differences in heart/body weight ratio were observed among the Con,Con+IMC,and Ade+IMC groups.These data suggested that adenine diet feeding induced heart hypertrophy in mice,and IMC supplementation completely blocked it.FGF23 is a bone-derived hormone that has been shown to cause left ventricular hypertrophy.We tested the circulating levels of FGF23 in mice in each group.The results showed that compared with the Con group,the circulating levels of FGF23 in the Ade group increased significantly,while the circulating levels of FGF23 in the Ade+IMC group were significantly lower than those in the Ade group.The decrease indicated that supplementation of IMC can significantly reduce circulating FGF23 levels caused by adenine feeding,which partially explains the improvement of heart hypertrophy in the Ade+IMC group.10 Inhibition of TMAO production ameliorated dyslipidemia in CKD micePlasma lipids were measured in these 4 groups of mice.Adenine diet feeding worsened the hyperlipidemia phenotypes in apoE knockout mice with increased plasma TG,TC,and VLDL/IDL/LDL-C levels as compared to those of the Con and Con+IMC groups.The Ade+IMC group exhibited significant decreases in plasma TC and VLDL/IDL/LDL-C levels as compared to the Ade group,suggesting improved plasma lipoprotein profile.11 Effect of inhibiting TMAO production on vascular diseases in CKD miceThe atherosclerotic lesion area at the aortic root was carefully quantified.Unexpectedly,there were no significant differences in mean atherosclerotic lesion area at the aortic root region among the 4 groups of mice.The calcification area at the aortic root was quantified too and there were no significant differences in the mean calcification area either.These results suggest that adenine diet feeding didn't cause the aggravation of atherosclerosis or calcification.Therefore,it is not possible to observe the effect of inhibiting TMAO production on atherosclerosis or calcification in this CKD mouse model.The aortic RNA of each group of mice was extracted for quantitative RT-PCR.The results showed that compared with the Con group and Con+IMC group,the expression levels of inflammation genes Ccl2,Cox2,116,and Vcam1 in the aorta of the Ade group and Ade+IMC group were significantly reduced,indicating that adenine itself can significantly reduce local inflammation in the aorta of mice.Adenine is a substrate of adenosine,and adenosine receptors play an important role in inhibiting vascular inflammation.We tested the expression level of adenosine receptor genes in the mice aorta and found that compared with the Con group,the expression of A2B adenosine receptor in the aorta of the Ade group had significant changes,suggesting that adenosine-adenosine receptor pathway may be involved in the protective process of adenine against atherosclerosis.12 Effect of extending adenine feeding on atherosclerosis in CKD miceIn animal experiment 2,we extended the adenine feeding period to 24 weeks.The survival curve showed that the mice in the Con group did not die during the entire experimental period,and half of the mice in the Ade group died at the end of the experiment.From the 12th week,the body weight of the two groups of mice began to differ,and the body weight of the Ade group was significantly lower than that of the Con group.At the end of the experiment,the levels of plasma creatinine,urea,cystatin C and TMAO in the Ade group were significantly higher than those in the Con group,and their plasma levels of TNF-? and IL6 were also significantly higher,suggesting that adenine feeding aggravated the inflammatory state of apoE knockout mice.The atherosclerotic lesion at the aortic root of mice was detected and quantitatively analyzed again.The results showed that there was still no significant difference in the mean atherosclerotic lesion area at the aortic root region between the Con group and the Ade group.13 Effect of extending adenine feeding on heart hypertrophy in CKD miceAt the end of the experiment,the mice were examined by echocardiography.The results showed that compared with the Con group,the IVS,LVID,LV Vol,LV mass and LV mass/Body weight of the Ade group increased significantly.The LVPW had a trend to increase but was not statistically significant yet.There was no significant difference in LVEF between the two groups.After the mice were sacrificed,the actual heart weight was measured,and the heart/body weight ratio was calculated,which was significantly higher in the Ade group than that in the Con group.The above results confirmed again that there is complication of heart hypertrophy in adenine-induced CKD mouse model.Since the thickness of ventricular wall and the diameter of ventricular cavity increased almost in the same proportion,the heart hypertrophy in this model may be the eccentric hypertrophy.Conclusion(1)The plasma TMAO levels were increased in the adenine-induced CKD mouse model.Inhibition of TMAO production by IMC can reduce renal inflammation and fibrosis and improve renal function in CKD mice;(2)In vitro experiments confirmed that TMAO could induce inflammation by activating NF-?B pathway in renal epithelial cells.Macrophage inflammation may also be involved in the process of kidney damage caused by TMAO;(3)Heart hypertrophy was found in adenine-induced CKD mice.Inhibition of TMAO production by IMC could significantly attenuate heart hypertrophy and alleviate CKD related cardiovascular damage.