Ribose and cellular defense mechanisms -
We next addressed the mechanism of how uridine supports the growth of UPP1 -expressing cells. Previous studies have noted the beneficial effect of uridine in the absence of glucose and proposed mechanisms that include the salvage of uridine for nucleotide synthesis and its role in glycosylation 4 , 5 , 6 , 7 , 8.
Others reported the beneficial role of uridine phosphorylase in maintaining ATP levels and viability during glucose restriction in the brain 9 , 10 , To further investigate the molecular mechanism of uridine-supported proliferation, we performed a secondary genome-wide CRISPR—Cas9 depletion screen using K cells expressing UPP1 -FLAG grown on glucose or uridine Fig.
and are corrected for natural isotope abundance. g , Schematic of uridine-derived ribose catabolism integrating gene essentiality results in glucose versus uridine. Gln, glutamine; Asp, aspartate.
We found that, although most essential gene sets were shared between glucose and uridine conditions, three major classes of genes were differentially essential in uridine as compared to glucose Fig.
Accordingly, uridine-grown cells were particularly sensitive to depletion of PGM2 , TKT and RPE , or to TKT inhibition, while they were insensitive to the de novo pyrimidine synthesis inhibitor brequinar Fig. In contrast, genes of the oxidative branch of the PPP G6PD , PGLS , PGD did not score differentially between glucose and uridine.
Central enzymes of glycolysis were essential both in glucose and in uridine, indicating that a functional glycolytic pathway is required for survival with uridine alone. However, our comparative analysis revealed that several upper glycolytic enzymes encoded by ALDOA , GPI and HK2 were dispensable in uridine, and only essential in glucose Fig.
Not all steps of upper glycolysis scored in either condition, potentially due to the multiple genes with overlapping functions encoding glycolytic enzymes, a common limitation in single gene-targeting screens. The essentiality of the non-oxPPP, with the dispensability of upper glycolysis in uridine Fig.
Lactate secretion and glycolytic utilization of uridine, however, were excluded in earlier work 4 , 5 , 6 , 7 , 8. Nonetheless, given the importance of PPP enzymes and the dispensability of upper glycolysis, we reinvestigated this possibility and measured lactate secretion in uridine-grown cells.
Strikingly, we found that UPP1 -expressing cells grown in uridine secreted high amounts of lactate Fig. Accordingly, we found using liquid chromatography—mass spectrometry LC—MS that uridine restored steady-state abundance of most central carbon metabolism detected in the absence of glucose, strongly suggesting some degree of lower glycolysis activity from uridine Extended Data Fig.
To directly test if uridine-derived ribose could serve as a substrate for glycolysis, we designed a tracer experiment using isotopically labelled uridine with five ribose carbons 13 C 5 -uridine and LC—MS Fig. UPP1 -expressing cells avidly incorporated 13 C 5 -uridine, as seen by the presence of 13 C in all the intracellular intermediates of the PPP and glycolysis analysed, including ribose-phosphate, upper and lower glycolytic intermediates and lactate, while control cells showed very little label incorporation.
As in cell lines, we found 13 C incorporation in ribose-phosphate and glycolysis in 13 C 5 -uridine-treated animals Fig. Incorporation efficiency was smaller than in cell culture, as expected from low-dose 13 C 5 -uridine injection, shorter treatment time and competition with other endogenous substrates in vivo, including unlabelled uridine.
We also found modest but significant incorporation of uridine-derived 13 C in glucose, indicating gluconeogenesis from uridine-derived carbons Fig. Together, our results indicate that in cell lines and in animals in vivo, uridine catabolism provides ribose for the PPP, and that the non-oxPPP and the glycolytic pathway communicate via F6P and G3P to replenish glycolysis thus entirely bypassing the requirement for glucose in supporting lower glycolysis, biosynthesis and energy production in sugar-free medium Fig.
We next sought to determine whether any human cell lines exhibit a latent ability to use uridine-derived ribose to grow on uridine when glucose is absent without the need for over-expression.
Cells from the melanoma and the glioma lineages grew remarkably well in uridine as compared to the other lineages, whereas Ewing sarcoma cells grew significantly less well Fig. Cell lines from the PRISM collection have been extensively characterized at a molecular level 14 , so we searched for genomic factors that correlate with the ability to grow on uridine Supplementary Table 1.
Genome wide, the top-scoring transcript, protein and genomic copy number variant was UPP1 Fig. Expression of UPP1 across the CCLE collection was the highest in cell lines of skin origin Extended Data Fig. In agreement with these results, we confirmed significant, UPP1 -dependent, proliferation and uridine catabolism in melanoma cells grown in sugar-free medium supplemented with uridine or RNA Fig.
We conclude that the endogenous expression of UPP1 is necessary and sufficient to support the growth of cancer cells on uridine. False discovery rates FDRs were calculated using a Benjamini—Hochberg algorithm correcting for multiple comparisons UPP1 is encoded on Chr7p MDA, MDA-MBS.
with two-sided t -test relative to untreated cells. We next investigated the factors that promote UPP1 expression and growth on uridine by integrating our results with CCLE data to prioritize transcription factors, which highlighted MITF as a strong candidate in melanoma cells, both at the protein and the transcript level Fig.
We found that MITF over-expression promoted UPP1 expression and uridine growth Extended Data Fig. Accordingly, siRNA-mediated depletion of MITF decreased UPP1 expression in melanoma cells Extended Data Fig. Our solid tumour PRISM cancer cells collection did not include cells of the immune lineage, where UPP1 is expressed at high levels 17 , 18 , so we asked whether immune cells exhibit the capacity to metabolize ribose from uridine either at baseline or in a transcriptionally regulated manner.
Among the immunostimulatory molecules, RNA enhanced UPP1 expression, suggesting the existence of a feed-forward loop, where RNA and conceivably RNA-containing pathogens and debris may trigger UPP1 expression and uridine salvage for building blocks and energy production.
Label incorporation from uridine ribose was also strongly increased in citrate and lactate after differentiation of THP1 and after BMDM stimulation with R, while it wasn't further increased in M-CSF-matured PBMCs, possibly due to high baseline capacity for uridine catabolism in these cells Fig.
Together, our results indicate that macrophages have the capacity to use uridine-derived ribose for glycolysis, and that UPP1 expression and uridine catabolism can sharply increase during cellular differentiation and in response to immunostimulating molecules, with cell type and species differences.
We next sought to determine whether glycolysis from uridine is under acute regulation in the same way as from glucose. Active OXPHOS tends to keep glucose uptake and glycolysis at lower levels, while acute inhibition of OXPHOS leads to an immediate and strong increase in glucose-supported glycolysis, as evidenced by a robust increase in the extracellular acidification rate ECAR following oligomycin treatment Fig.
Strikingly, we found no ECAR stimulation by OXPHOS inhibitors, no difference in 13 C 5 -uridine incorporation following antimycin blockage of the electron transport chain, and no increase in uridine import in OXPHOS-inhibited UPP1 -expressing cells grown on uridine Fig. Because glycolysis from both uridine and glucose share a common pathway from G3P Fig.
Consistent with this notion, we observed no stimulation of ECAR in mannose-grown cells, a sugar connected to glycolysis by F6P Extended Data Fig.
We conclude that substrates such as uridine can enter glycolysis in a constitutive way, in contrast to glucose, by bypassing regulatory steps of upper glycolysis such as glucose transport and initial phosphorylation.
a , Schematic of glycolysis inhibition by OXPHOS. G6P, glucosephosphate. O, oligomycin; C, CCCP; A, antimycin A. In line with this, we next performed a competition experiment to evaluate if the presence of glucose affects the incorporation of uridine in cells.
Incorporation of uridine in lactate was notably not affected by competition with glucose in our experimental conditions, despite the presence of a large molar excess of glucose Fig.
Therefore, and in agreement with a bypass of regulatory steps of upper glycolysis, uridine can be incorporated into cells even when lactate production from glucose is saturated, suggesting constitutive import and catabolism. Cells with severe OXPHOS dysfunction classically have to be grown on glucose, and uridine must be supplemented 1.
The traditional explanation has been that glucose is required to support glycolytic ATP production as OXPHOS is debilitated, and that uridine supplementation is required for pyrimidine salvage given that de novo pyrimidine synthesis via DHODH requires coupling to a functional electron transport chain 1 , 3 Extended Data Fig.
Having observed energy harvesting from uridine, we finally tested whether uridine-derived ribose could also benefit OXPHOS-inhibited cells in the absence of glucose. We found a significant UPP1 -dependent rescue of viability in galactose-grown cells treated with antimycin A Fig.
For decades it has been known that cells with mitochondrial deficiencies are dependent on uridine to support pyrimidine synthesis given the dependence of de novo pyrimidine synthesis on DHODH, whose activity is coupled to the electron transport chain 1.
Although it has been documented, it is less appreciated that uridine supplementation can support cell growth in the absence of glucose 4 , 5 , 6 , 7 , 8 , 9 , Here, we show that, in addition to nucleotide synthesis, uridine can serve as a substrate for energy production, biosynthesis and gluconeogenesis.
By comparing uridine to other nucleosides and using similar tracer experiments to ours, Wice et al. However, they did not detect pyruvate and lactate in uridine, and concluded that uridine does not participate in glycolysis, but rather is required for nucleotide synthesis, and proposed that energy is derived exclusively from glutamine in the absence of glucose 6 , 7.
Loffler et al. and Linker et al. reached the same conclusion 4 , 8. Our observations based on a genome-wide CRISPR—Cas9 screening and metabolic tracers Fig.
It has previously been reported that uridine protects cortical neurons and immunostimulated astrocytes from glucose deprivation-induced cell death, in a way related to ATP, and it was hypothesized that uridine could serve as an ATP source 9.
Our genetic perturbation and tracer studies are consistent with this hypothesis. The capacity to harvest energy and building blocks from uridine appears to be widespread. Here, we report very high capacity for uridine-derived ribose catabolism in melanoma and glioma cell lines Fig. Based on gene expression atlases 18 , 19 , we predict uridine may be a meaningful source of energy in blood cells, lung, brain and kidney, as well as in certain cancers.
Uridine is the most abundant and soluble nucleoside in circulation 20 and it is possible that uridine may serve as an alternative energy source in these tissues, or for immune and cancer metabolism, similar to what has been proposed for other sugars and nucleosides 21 , 22 , It is notable that the strongest human metabolic quantitative trait loci for circulating uridine corresponds to UPP1 ref.
A fascinating aspect of glycolysis from uridine is its apparent absence of regulation, at least at shorter timescales. The ability of uridine to serve as a constitutive input into glycolysis might have clinical implications for human diseases, as uridine is present at high levels in foods such as milk and beer 26 , 27 , and previous in vivo studies have shown that a uridine-rich diet leads to glycogen accumulation, gluconeogenesis, fatty liver and pre-diabetes in mice 28 , We now report that glycolysis from uridine lacks at least two checkpoints as 1 it is not controlled by OXPHOS Fig.
Although glycolysis from uridine appears to occur at a slower pace than from glucose, we speculate that constitutive fuelling of glycolysis and gluconeogenesis from a uridine-rich diet may contribute to human conditions such as fatty liver disease and diabetes.
This ability of uridine to bypass upper glycolysis may be beneficial in certain cases. At longer timescales, UPP1 expression and capacity for ribose catabolism from uridine appear to be determined by cellular differentiation and further activation by extracellular signals. Here we focused on the monocytic lineage and found that 1 in THP1 cells, UPP1 expression and activity sharply increased during differentiation and polarization, 2 high baseline rates of glycolysis from uridine are observed in M-CSF-matured PBMCs and 3 treatment with immunostimulating molecules acutely promote both UPP1 expression and uridine catabolism in BMDMs Fig.
It is thus likely that NF-κB may serve as a transcription factor for UPP1. Supporting this assertion, we found that blocking NF-κB signalling with upstream IKK inhibitors abolished Rinduced Upp1 expression Extended Data Fig. Uridine phosphorylase and ribose salvage by UPP1 appears to lie downstream of a number of signalling pathways with potential relevance to disease.
We have demonstrated that uridine breakdown is promoted by MITF, a transcription factor associated with melanoma progression, which we show binds upstream of UPP1 to promote its expression Extended Data Fig.
In an accompanying study, Nwosu, Ward et al. It is notable that both MITF and NF-kB can act downstream of KRAS—MAPK 34 , 35 , 36 , 37 , 38 and that some pancreatic cell lines with high uridine phosphorylase activity highlighted by Nwosu, Ward et al. Finally, we found that RNA in the medium can replace glucose to promote cellular proliferation Fig.
Recycling of ribosomes through ribophagy, for example, plays an important role in supporting viability during starvation Cells of our immune system also ingest large quantities of RNA during phagocytosis, and we experimentally showed that the expression of UPP1 increases with macrophage activation Fig.
Uridine seems to be the only constituent of RNA that can be efficiently used for energy production, at least in K cells Fig. Whereas the salvage of RNA to provide building blocks during starvation has long been appreciated for nucleotide synthesis, to our knowledge, its contribution to energy metabolism has not been considered in the past, except for some fungi that can grow on minimum media with RNA as their sole carbon source We speculate that, similar to glycogen and starch, RNA itself may constitute as large stock of energy in the form of a polymer, and that it may be used for energy storage and to support cellular function during starvation, or during processes associated with high energy costs such as the immune response.
K CCL , T CRL , HeLa CCL-2 , A CRL , A CRL , SH4 CRL , MDA-MBS HTB , SK-MEL-5 HTB , SK-MEL HTB and THP1 TIB cell lines were obtained from the American Type Culture Collection ATCC.
UACC, UACC and LOX-IMVI cells were obtained from the Frederick Cancer Division of Cancer Treatment and Diagnosis DCTD Tumor Cell Line Repository.
All cell lines were re-authenticated by STR profiling at ATCC before submission of the manuscript and compared to ATCC and Cellosaurus ExPASy STR profiles in , with the exception of THP1 TIB and U CRL Cells lines from the PRISM collection were obtained from The PRISM Lab Broad Institute and were not further re-authenticated.
MDA-MBS cells were previously assumed to be ductal carcinoma cells and recent gene expression analysis assigned them to the melanoma lineage ATCC. All growth assays, metabolomics, screens and bioenergetics experiments were performed in medium containing dialysed FBS.
For RNA and other nucleoside complementation assays, 0. Cell viability in glucose and galactose was determined using the same Vi-Cell Counter assay. Measurements were taken from distinct samples. For ORF screening, K cells were infected with a lentiviral-carried ORFeome v8.
Cells were infected at a multiplicity of infection of 0. Barcode sequencing, mapping and read count were performed by the Genome Perturbation Platform Broad Institute. For screen analysis, log 2 normalized read counts were used, and P values were calculated using a two-sided t -test.
The presence of lentiviral recombination within the ORFeome library was not tested and as such genes that dropped out should be considered with caution, as these may represent unnatural proteins Twenty-four hours after infection, cells were selected with 0. Protein concentration was determined from total cell lysates using a DC protein assay Bio-Rad.
Gel electrophoresis was done on Novex Tris-Glycine gels Thermo Fisher Scientific before transfer using the Trans-Blot Turbo blotting system and nitrocellulose membranes Bio-Rad.
Washes were done in TBST. Specific primary antibodies were diluted at a concentration of —, in blocking buffer. Fluorescent-coupled secondary antibodies were diluted at a ratio of , in blocking buffer. Membranes were imaged with an Odyssey CLx analyzer Li-cor with Image Studio Lite v4.
The following antibodies were used: FLAG M2 Sigma, F , Actin Abcam, ab , TUBB Thermo, MA , UPP1 Sigma, SAB , MITF Sigma, HPA , TYR Santa Cruz sc , MLANA CST, , HK2 CST, , GPI CST, , ALDOA CST, , TKT CST, , RPE Proteintech, AP , PGM2 Proteintech, AP , UCK2 Proteintech, AP , TYMS Proteintech, AP , S6 ribosomal protein Santa Cruz, sc and phosphor-S6 Santa Cruz, sc Two commercially available antibodies to UPP2 were tested Sigma, SAB; Abcam, ab , but no specific band could be detected.
The medium was replaced with fresh medium on days 3 and 5. On day 6, all wells reached confluency and cells were lysed. Barcode abundance was determined from sequencing, and unexpectedly low counts for example, from sequencing noise were filtered out from individual replicates so as not to unintentionally depress cell line counts in the collapsed data.
Replicates were then mean-collapsed, and log fold change and growth rate metrics were calculated according to equations 1 and 2 :. where n u and n g are counts from the uridine and glucose supplemented conditions, respectively, n 0 and n f are counts from the initial and final timepoints, respectively, and t is the assay length in days.
Data analysis and correlation analysis were performed by The PRISM Lab following a published workflow qPCR was performed using the TaqMan assays Thermo Fisher Scientific.
Human PBMCs and mouse BMDM data were normalized to TBP , and liver mouse data were normalized to Rplp2 , both using the ΔΔCt method. qPCR primers for ChIP are described below.
Fixation was stopped by adding glycine final concentration of 0. Cells were harvested by scraping with ice-cold PBS.
Samples were centrifuged to remove debris and diluted tenfold in immunoprecipitation dilution buffer DNA was purified with QIAquick PCR purification kit Qiagen. Purified DNA was co-transfected with a GFP-expressing plasmid in the cell lines of interest using Lipofectamine Thermo Fisher Scientific.
UPP1 depletion in single-cell clones was assessed by protein immunoblotting using antibodies to UPP1. The 9-bp deletion in clone 2 is expected to produce a truncated protein hypomorphic allele.
Three hours after plating, cells were further treated with 0. Human PBMCs were isolated from buffy coats of blood donors from a local transfusion centre.
On day 6, cells were detached, counted and replated at 1. PBMC polarization was performed as with BMDMs. A secondary genome-wide CRISPR—Cas9 screening was performed using K cells expressing UPP1 -FLAG and a lentiviral-carried Brunello library Genome Perturbation Platform, Broad Institute containing 76, sgRNAs 44 , in duplicate.
Cells were infected with multiplicity of infection of 0. DNA isolation was performed as for the ORFeome screen. The log 2 fold change of each sgRNA was determined relative to the pre-swap control.
For each gene in each replicate, the mean log 2 fold change in the abundance of all four sgRNAs was calculated. log 2 fold changes were averaged by taking the mean across replicates.
For each treatment, a null distribution was defined by the 3, genes with lowest expression. To score each gene within each treatment, its mean log 2 fold change across replicates was z -score transformed, using the statistics of the null distribution defined as above.
Cells were incubated for five additional hours before metabolite extraction. All animal experiments in this paper were approved by the Massachusetts General Hospital, the University of Massachusetts Institutional Animal Care and Use Committee, or the Swiss Cantonal authorities, and all relevant ethical regulations were followed.
All cages were provided with food and water ad libitum. Food and water were monitored daily and replenished as needed, and cages were changed weekly.
A standard light—dark cycle of h light exposure was used. Animals were housed at 2—5 per cage. Liver was flash frozen in liquid nitrogen before subsequent analysis, and blood was collected in EDTA plasma tubes, spun and plasma was stored for further analysis.
For each run, the total flow rate was 0. Data were acquired using Xcalibur v. Data were analysed using TraceFinder v. The flow rate was then increased to 0. Approximately 1. FBS was omitted. Data were analysed using the Seahorse Wave Desktop Software v.
Data were not corrected for carbonic acid derived from respiratory CO 2. Lactate secretion in the culture medium was determined using a glycolysis cell-based assay kit Cayman Chemical. Cells were then re-counted and seeded in fresh medium of the same formulation and incubated for three additional hours.
Cells were then spun down and lactate concentration was determined on the supernatants spent media. Gene Ontology GO analysis was performed using GOrilla with default settings and using a ranked gene list as input The complete unfiltered data can be found in Supplementary Table 1.
cDNAs of interest were custom designed Genewiz or IDT and cloned into pWPI-Neo or pLV-lenti-puro using BamHI and SpeI New England Biolabs. All reported sample sizes n represent biological replicate plates or a different mouse.
All attempts at replication were successful. Statistical tests were performed using Microsoft Excel and GraphPad Prism 9. Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
All data generated or analysed during this study are included in the article and its Supplementary Information. Results of the ORFeome, the CRISPR—Cas9 and the PRISM screens are available in Supplementary Table 1. Source data are provided with this paper.
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Mice were acclimatized to the housing environment for at least 1 week before the experiments. On the day protocol was completed, blood samples were taken for measurement of fasting blood glucose with OneTouch Ultra2 blood glucose meter LifeScan Europe, Switzerland.
Then, mice were sacrificed under mild ethyl ether anesthesia and their kidneys were harvested. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of the Virginia Commonwealth University.
A conditionally immortalized mouse podocyte cell line Graciously provided by Dr. Klotman, Division of Nephrology, Department of Medicine, Mount Sinai School of medicine, New York, NY, United States , were cultured and maintained as described before Abais et al. For all experiments, culture medium was replaced with serum-free medium for 24 h prior to treatments.
Podocytes were incubated with 25 mM D-ribose Sigma, United States , 25 mM L-ribose AK scientific, United States as negative control AK scientific, United States and 25 mM D -glucose Sigma, United States as positive control for 24 h.
To inhibit the role of AGEs, AGEs formation inhibitor aminoguanidine AG, 50 μM, Sigma Aldrich and a breaker of AGEs-based cross-links, alagebrium chloride ALT, μM, TCI AMERICA were used 30 min prior to treatments Dhar et al.
Glomerular morphology was observed and assessed semi-quantitatively as described previously Raij et al. Total urinary protein concentrations were determined spectrophotometrically using Bradford assay Sigma, United States.
Then slides were incubated with biotinylated secondary antibodies and a streptavidin peroxidase complex PK, Vector Laboratories, Burlingame, CA, United States. Finally, samples observed with microscopy as described previously Raij et al. The area percentage of the positive staining was calculated with Image Pro Plus 6.
After treatments, kidney slides and podocyte culture coverslips were fixed, blocked and incubated with primary antibodies against NLRP3 , Abcam, Cambridge, MA, United States , ASC , Santa Cruz Biotechnology, Dallas, TX, United States , cleaved-caspase-1 , Santa Cruz Biotechnology, Dallas, TX, United States , podocin , Sigma , or desmin , Thermo Fisher Scientific at 4°C overnight.
Then slides were incubated with corresponding second antibodies with either Alexa- or Alexalabeled Invitrogen, Carlsbad, CA, United States. For example, slides incubated with NLRP3 were then incubated with donkey anti goat secondary antibody, Alexa fluor plus , slides incubated with ASC, cleaved-caspase-1 or Podocin were then incubated with donkey anti mouse secondary antibody, Alexa fluor plus , slides incubated with desmin were then incubated with donkey anti rabbit secondary antibody, Alexa fluor plus After that, slides were observed with a laser scanning confocal microscope Fluoview FV, Olympus, Japan.
Co-localization coefficient was analyzed with Image Pro Plus 6. Equivalent amount of proteins 20—30 μg was resolved on SDS-PAGE gels and transferred to PVDF membrane.
After blocking, membranes were probed with primary antibodies rabbit anti-Cle-Caspase-1 , cell signaling technology , rabbit anti-pro-Caspase-1 , Abcam, Cambridge, MA and rabbit anti-β-actin , Santa Cruz Biotechnology, Dallas, TX, United States at 4°C overnight.
After incubated with donkey anti-rabbit-HRP IgG , Santa Cruz Biotechnology, Dallas, TX, United States , immunoreactive bands were detected by chemiluminescence techniques with LI-COR Odyssey Fc. The intensity of the specific bands was calculated with ImageJ software NIH, Bethesda, MD, United States.
RAGE small interference RNAs siRNAs was purchased from Santa Cruz Biotechnology, Dallas, TX, United States. Data are presented as means ± SE. We first tested whether D-ribose treatment induced podocyte injury and glomerular damage in WT and Asc gene knockout mice.
As shown in Figures 1A,B , i. injection of D-ribose for 30 days induced proteinuria and albuminuria in WT mice in comparison with mice treated with vehicle, which were markedly blocked by simultaneous administration of AG, an AGEs formation inhibitor.
In Asc gene knockout mice, D-ribose-induced proteinuria and albuminuria were much less severe. Consistently, WT mice receiving D-ribose injection had remarkable extracellular matrix and collagen deposition, mesangial cell expansion and capillary collapse, indicating a typical glomerular sclerotic pathology Figures 1C,E , which were abolished by AG co-treatment.
Using confocal microscopy, D-ribose was found to markedly alleviate podocin staining, but increase the fluorescence intensity of desmin staining Figures 1D,F in glomeruli of WT mice. After injection of D-ribose for 30 days, fasting-blood glucose in D-ribose-treated mice was significantly decreased compared to Vehl group All these glomerular and podocyte injuries induced by D-ribose were substantially blocked by administration of AG.
Additionally, in Asc gene knockout mice, D-ribose treatment failed to induce podocyte injury and glomerular damage, as shown by no differences in all measured glomerular injurious parameters between mice receiving Vehl and D-ribose treatments. Figure 1. D-ribose induced glomerular dysfunction and injury in an AGEs-dependent manner.
Vehl, Vehicle; D-R, D-ribose; AG, Aminoguanidine. As depicted in Figure 2A , the confocal microscopic analysis demonstrated that D-ribose remarkably increased co-localization of NLRP3 with ASC or caspase-1 increased yellow staining in glomeruli compared with Vehl group, which was blocked by AG treatment.
Quantitation of the NLRP3 co-localization shows that NLRP3 inflammasome formation only occurred in mice receiving D-ribose, but not in mice of Vehl group Figure 2B. Based on our previous studies showing that NLRP3 molecules are most enriched in podocytes, these results indicate the formation of NLRP3 inflammasome in glomerular podocytes of mice receiving D-ribose.
Consistently, IL-1β as a prototype inflammatory cytokine produced by the NLRP3 inflammasome also significantly increased in glomeruli of mice treated with D-ribose compared to mice in Vehl group, which was markedly attenuated by AG Figures 2C,D.
In Asc gene knockout mice, however, D-ribose failed to induce NLRP3 inflammasome formation and activation in glomeruli. These findings demonstrate that NLRP3 inflammasome activation and consequent IL-1β production may contribute to D-ribose-induced podocyte injury and glomerular sclerosis.
Figure 2. D-ribose-induced NLRP3 inflammasome formation and activation in glomeruli. To explore the mechanisms by which D-ribose induces NLRP3 inflammasome activation and podocyte injury, we observed changes in AGEs formation and RAGE expression in mouse glomeruli.
By immunohistochemistry, AGEs and RAGE were remarkably elevated in glomeruli of mice receiving D-ribose compared to Vehl group, and it could be totally blocked by AG, the AGEs formation inhibitor Figures 3A,B. Figure 3. Enhancement of AGEs production and RAGE expression in glomeruli of D-ribose-treated mice.
To further explore the mechanisms by which D-ribose activates NLRP3 inflammasome, we performed in vitro experiments in cultured podocytes. Caspase-1 inhibitor, YvAD, was used to pre-treat podocytes to test whether D-ribose indeed activates the NLRP3 inflammasome in podocytes in comparison with its isoform, L-ribose and positive control, D -glucose.
By using confocal microscopy, we demonstrated that co-localization of NLRP3 green with ASC red or caspase-1 red , an indicative of NLRP3 inflammasome formation, was much higher in podocytes treated with D-ribose than Vehl and L-ribose, but similar to podocytes treated with D -glucose.
However, prior treatment of podocytes with YvAD almost completely abolished the enhanced co-localization of NLRP3 with ASC or caspase-1 induced by D-ribose or D -glucose Figures 4A,B. The co-localization coefficient was summarized and is shown below the representative confocal microscopic images Figures 4C,D.
Figure 4. Effects of D-ribose on NLRP3 inflammasome formation and activation in podocytes. Ctrl, control; Vehl, Vehicle; L-R, L-ribose; D-R, D-ribose; D-G, D -glucose.
Based on previous studies, increased cleavage of caspase-1 and enhanced caspase-1 activity and consequent IL-1β production reflect NLRP3 inflammasome activation. The present study found that D-ribose treatment remarkably increased the level of cleaved caspase-1 22 kDa , but not procaspase-1 51 kDa Figures 4E,F , caspase-1 activity Figure 4G and IL-1β production Figure 4H , and prior treatment with YvAD almost completely attenuated these changes.
Additionally, we demonstrated that D -glucose had D-ribose-like effect on NLRP3 inflammasome activation in podocytes. To further test whether AGEs play an important role in the formation and activation of NLRP3 inflammasome induced by D-ribose, AG, an AGEs formation inhibitor and alagebrium chloride ALT , an AGEs breaker were used prior to incubation with D-ribose or vehicle.
As shown in Figure 5 , co-localization of NLRP3 with ASC Figures 5A,C or caspase-1 Figures 5B,D was much higher in D-ribose-treated podocytes than Vehl- or L-ribose treated podocytes, but similar to D -glucose-treated podocytes. However, prior treatment of podocytes with AG or ALT completely blocked D-ribose or D -glucose-induced co-localization of NLRP3 with ASC or caspase The level of cleaved caspase-1 Figures 5E,F , its activity Figure 5G and IL-1β production Figure 5H were remarkably elevated by D-ribose treatment, which was completely blocked by prior treatment of podocytes with AG or ALT.
AG or ALT was also found to attenuate D-ribose-induced increased expression of AGEs and RAGE in podocytes Figures 6A—D. D -glucose had a similar effect to D-ribose on NLRP3 inflammasome activation in podocytes, which was blocked by prior treatment of AG or ALT.
Figure 5. Effects of AGEs formation inhibition and their crosslink breakers on D-ribose-induced NLRP3 inflammasome formation and activation in podocytes. AG, aminoguanidine. ALT, alagebrium chloride.
Figure 6. AG and ALT blocked D-ribose-induced AGEs production and RAGE overexpression in podocytes. We further examined the role of RAGE in D-ribose-induced NLRP3 inflammasome formation and activation.
In these experiments, a siRNA against RAGE siRAGE was transfected to podocytes prior to administration of D-ribose or controls. This siRNA effectively silenced RAGE expression and blocked D-ribose-induced increase in RAGE expression as shown by Western blot analysis Figures 7A—C.
By confocal microscopy, co-localization of NLRP3 green with ASC red or caspase-1 red was demonstrated to be much higher in D-ribose-treated podocytes than Vehl- or L-ribose-treated podocytes.
RAGE gene silencing completely blocked D-ribose-induced co-localization of NLRP3 with ASC or caspase-1 Figures 8A—D. RAGE gene silencing markedly diminished D-ribose-induced increase in cleaved caspase-1 levels Figures 8E,G , caspase-1 activity Figure 8F and IL-1β production Figure 8H.
Similarly, D -glucose-induced NLRP3 inflammasome formation and activation in podocytes were also attenuated by RAGE gene silencing. Figure 7. RNA interference of RAGE prevented D-ribose-induced NLRP3 inflammasome formation and activation in podocytes.
Figure 8. The present study was designed to determine whether D-ribose induces NLRP3 inflammasome activation and results in podocyte and glomerular injuries and whether it depends upon AGEs-RAGE signaling pathway. It was found that D-ribose induced NLRP3 inflammasome formation and activation, which led to podocyte injury and glomerular sclerosis in WT mice.
Knockout of gene encoding Asc blocked D-ribose-induced NLRP3 inflammasome activation and consequent podocyte injury in mouse glomeruli. Furthermore, in vitro studies using cultured podocytes showed that D-ribose indeed stimulated NRLP3 inflammasome formation and activation.
Inhibition of AGEs formation, increases in AGEs cleavage or silencing of RAGE gene all prevented D-ribose-induced NLRP3 inflammasome formation and activation, which protected podocytes from D-ribose-induced injury. These findings suggest that activation of RAGE by AGEs plays a crucial role in NLRP3 inflammasome activation and podocyte injury induced by D-ribose.
Diabetes mellitus is characterized by high blood glucose levels due to either insulin deficiency or insulin resistance, which generates many organ damages leading to degenerative complications such as diabetic retinopathy, and renal glomerular injuries.
Although increased blood D -glucose level is widely known to be responsible for the development of diabetic complications, D-ribose is now emerging as a novel pathogenic factor for organ damages during DM.
However, the molecular mechanism mediating the pathogenic action of D-ribose remains poorly understood. In the present study, we first demonstrated that i. injection of D-ribose induced podocyte injury and glomerular sclerosis in mice, which were associated with NLRP3 inflammasome activation and consequent increase in IL-1β production, suggesting that NLRP3 inflammasome activation may be a critical mechanism for D-ribose-induced podocyte injury and progressive development of glomerular sclerosis.
In Asc gene knockout mice, D-ribose failed to produce NLRP3 inflammasome activation, which prevented D-ribose-induced podocyte injury and glomerular sclerosis.
This further confirms that NLRP3 inflammasome activation is crucial for the pathogenic action of D-ribose. To our knowledge, these findings provide the first experimental evidence that D-ribose may induce podocyte injury and glomerular sclerosis via activation of the NLRP3 inflammasome.
Recently, it has been reported that NLRP3 inflammasome activation serves as a triggering mechanism leading to glomerular injury and ultimate end-stage renal disease ESRD under different pathological conditions Yi et al. In this regard, there is evidence that the expression of NLRP3 inflammasome molecules and pro-inflammatory cytokines increased in patients and mice suffering from T2DM Vandanmagsar et al.
NLRP3 or caspase-1 gene knockout and caspase-1 inhibition blocked or even reversed the progression diabetic nephropathy in mice Wen et al. All these results suggest that the NLRP3 inflammasome may be an important pathogenic factor for the development of diabetic nephropathy, which is probably a new target for treatment of DN complications in the kidney Segelmark and Hellmark, Given that NLRP3 inflammasome activation may be initiated by the production of mitochondrial reactive oxygen species ROS and increased NADPH oxidase activity during DM Gao et al.
Degense levels are Avocado Rice Bowl to defenss increased in type II diabetes Riobse and increased blood D-ribose Alternate-day fasting for beginners involved in the development of diabetic mechanism such Avocado Rice Bowl diabetic Ribose and cellular defense mechanisms and Mecganisms. However, the mechanism mediating the ecllular role of D-ribose in nephropathy remains poorly understood. Administration of D-ribose daily for 30 days was found to induce NLRP3 inflammasome formation in glomerular podocyte, as shown by increased co-localization of NLRP3 with apoptosis-associated speck-like protein containing a caspase recruitment domain ASC or caspase This D-ribose-induced NLRP3 inflammasome formation was accompanied by its activation as evidenced by increased IL-1β production, a major product of NLRP3 inflammasome. Corresponding to NLRP3 inflammasome activation, D-ribose led to significant glomerular injury in mice.Video
Riboswitches Corrigendum: The Ribos Rest and recovery of PARPs Energy enhancement regulating defensd immune Avocado Rice Bowl. Poly adenosine diphosphate-ribose polymerases PARPs are a family of proteins defens for transferring ADP-ribose groups to target proteins to initiate the ADP-ribosylation, a highly conserved and fundamental post-translational modification in all defenes. PARPs play Rest and recovery roles mechanismms various cellular Rest and recovery, including Riobse chromatin structure, transcription, replication, recombination, and Xnd repair. Several studies have recently converged on the widespread involvement of PARPs and ADP-Ribosylation reaction in mammalian innate immunity. Innate immunity is an evolutionary conservative defense system that generates prompt immune responses to protect the host from tumorigeneses and pathogenic infections, which depends on the recognition of endogenous damage-associated molecular patterns DAMPs or exogenous conserved pathogen-associated molecular patterns PAMPs in many microorganisms by host pattern recognition receptors PRRsincluding four main families: Toll-like receptors TLRsretinoic acid-inducible gene I RIG-I -like receptors RLRscytosolic DNA sensor cyclic GMP-AMP synthase cGAS -stimulator of interferon genes STING axis, and the nucleotide-binding oligomerization domain NOD -like receptors NLRs. PARPs are the best-understood ARTs, and to date, 17 members of PARPs in humans have been identified.
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