Telomere maintenance mechanisms (TMM) are used by cancer cells to avoid apoptosis, 85–90% reactivate telomerase, while 10–15% use the alternative lengthening of telomeres (ALT). Due to anti-telomerase-based treatments, some tumors switch from a telomerase-dependent mechanism to ALT; in fact, the co-existence between both mechanisms has been observed in some cancers. Although different elements in the ALT pathway are uncovered, some molecular mechanisms are still poorly understood. Therefore, with the aim to identify potential molecular markers for the study of ALT, we combined in silico approaches in a 411 telomere maintenance gene set. As a consequence, we conducted a genomic analysis of these genes in 31 Pan-Cancer Atlas studies from The Cancer Genome Atlas and found 325,936 genomic alterations; from which, we identified 20 genes highly mutated in the cancer studies. Finally, we made a protein-protein interaction network and enrichment analysis to observe the main pathways of these genes and discuss their role in ALT-related processes, like homologous recombination and homology directed repair. Overall, due to the lack of understanding of the molecular mechanisms of ALT cancers, we proposed a group of genes, which after ex vivo validations, could represent new potential therapeutic markers in the study of ALT. Telomeres are nucleoprotein complexes that consist of a tandem 5’-TTAGGG-3’ sequence and protect the ends of eukaryotic chromosomes, preventing DNA damage response (DDR), end-to-end fusions and genomic instability . TelomerTRF1 and TRF2 associate directly with the TRF1 interacting nuclear factor 2 (TINF2 or TIN2), which stabilizes their bond with the telomere; the shelterin complex subunit, the telomerase recruiting factor (ACD or TPP1) and the TRF2 interacting protein (TRF2IP or RAP1) complete the complex that protects and regulates telomere structure, DNA damage response and lengthening . NonetheTo avoid senescence or apoptosis caused by telomere shortening, cancer cells take advantage of a set of mechanisms known as Telomere Maintenance Mechanisms (TMM), which include: telomerase reactivation and the alternative lengthening of telomeres (ALT) ,6. A higALT-positive (ALT+) cells display a characteristic phenotype. Telomeric DNA in ALT+ cells is constantly elongating, commonly depending on break-induced replication (BIR) processes, which are part of the homologous recombination (HR) pathway or homology-directed repair (HDR) mechanism ,11. ALT+The main characteristic of ALT+ telomeres is the interaction with promyelocytic leukemia (PML) proteins, which altogether are believed to function as platforms for telomere recombination and are called ALT-associated PML bodies (APBs) ,14. In fATRX, DAXX, H3F3A, or the BIR-related pathways dependent or independent of RAD51 and RAD52 [Although, many ALT-related genomic alterations have been resolved, such as the loss of function of nd RAD52 ,10,15, tnd RAD52 Many anti-telomerase-based cancer therapies are believed to cause the switching in some tumors from telomerase to ALT ,16. IndeEven though several ALT-associated factors have been uncovered, the pathway activation, regulation and functioning require further investigation . TherefoTo fulfill this need, we evaluated the genomic alterations of 411 telomere maintenance (TM) genes in 9282 samples from 31 Pan-Cancer Atlas (PCA) studies and applied different in silico approaches with the aim to identify and propose a set of genes which after ex-vivo validations could be used as potential molecular markers to improve the study of the ALT pathway.http://www.cancertelsys.org/telnet/) is a database that groups more than 2000 human TM genes. All genes are annotated according to their classification of TMM, TM function and a significance score given by the evidence of gene function in telomeres [TelNet  [p< 0.05) by using SPSS Statistics Software .Genomic alterations  were analyzed in the cBio Portal  ,21. A toal.org/) ,29,30,31The ALT mechanism is frequent in tumors of mesenchymal origin; however, ALT positive cells have been observed in epithelial tumors. Therefore, the prevalence of the ALT mechanism in the different TCGA cancer studies was reported as part of this study and the tumors were classified in three different categories: frequent ALT tumors, rare ALT tumors and not reported (for tumors without evidence of ALT+ cells). Additionally, the first quartile of the most altered genes from the PCA studies was classified in each of the categories described earlier.https://string-db.org/) was used. An interaction score of 0.9 (highest confidence) was set in the system configuration [p< 0.001) related to ALT were selected and differentiated by colors in the network.In order to predict the interactions among the ALT-related proteins, the STRING database  is a main process used by ALT+ cells to extend their telomeres , therefohttps://biit.cs.ut.ee/gprofiler/gost) [p< 0.001), based on Gene Ontology (GO), Molecular Function (MF), Biological Process (BP), KEGG, REACTOME database and Human Phenotype (HP).An enrichment analysis was made using the g:Profiler tool (er/gost) . The mosThe 411 genes from the TelNet database  Table S were filTo address the genomic alterations of ALT-related genes, these were analyzed in the cBioPortal ,21 by seA total of 325,936 genomic alterations were identified  and a pip > 0.01). As a result, it can be inferred that TM genes alterations and the ALT pathway across different types of tumors are not dependent on cancer staging.Finally, to better understand the implication of ALT-related genes in cancer progression, from primary tumors to metastasis, we analyzed the genomic alterations in each cancer stage b Table . Howevern = 103) was selected for further analysis and the highest frequencies of alterations for the 31 PCA studies were: UCS (56.054), OV (53.194), UCEC (49.387), ESCA (47.088), BLCA (44.883), ACC (44.658), SKCM (44.620), LUSC (43.822), STAD (43.005), BRCA (42.068), LUAD (40.942), CESC (40.116), COAD (39.038), SARC (37.418), LIHC (37.353), DLBC (37.256), HNSC (36.381), CHOL (35.444), PAAD (33.256), MESO (30.634), UVM (30.225), GBM (29.366), TGCT (29.132), KIRP (28.708), PRAD (28.258), LGG (27.387), PCPG (26.292), KIRC (24.168), LAML (21.255), THYM (20.185), and THCA (19.319)  a.TP53 (12.370), RAD21 (7.529), SENP5 (7.179), UBR5 (6.897), NSMCE2 (6.779), MCPH1 (6.332), PRKDC (6.091), RFC4 (5.662), RECQL4 (5.656), NBN (5.587), FBH1 (5.239), RAD1 (5.221), CCT5 (5.126), LRATD2 (5.013), RAD54B (4.969), ATR (4.940), SMG7 (4.888), TERF1 (4.817), ATF6 (4.778), and ARID1A (4.357).To further identify which genes displayed abnormal values of genomic alterations in the 31 PCA studies, a boxplot with these frequencies was constructed for the 411 ALT-related genes b. Hence,TP53 (9), UBR5 (8), RAD21 (6), SENP5 (6), NSMCE2 (5), RFC4 (5), MCPH1 (4), RECQL4 (4), ARID1A (3), CCT5 (3), LRATD2 (3), and RAD1 (3).Then, to obtain insights of the most altered genes in the PCA studies, additional boxplots were constructed c, along Finally, to elucidate which genomic alteration is most frequently observed across the 31 PCA studies, an oncoprint with the percentage of each alteration is shown for the first-quartile of genes d. As expThe presence of ALT in tumors across the 31 PCA studies have been analyzed and classified in three different categories: ALT frequent tumors, for cancers with ALT prevalence above the mean, ALT rare tumors, for cancers with prevalence below the mean, and tumors with no reports of the presence of ALT .n = 103) in the PCA studies in each ALT tumor group, a Venn diagram was constructed  were selected and marked with different colors in the nodes  [For our study, we found the highest frequency means of genomics alterations in the following PCA studies: UCS, OV, UCEC, ESCA, BLCA, ACC, SKCM, LUSC, STAD, and BRCA a. In addone H3.3 , and itsression) .TP53, additional ALT-related genes were identified and proposed in this study as possible molecular targets that have been predicted by Lovejoy et al. 2012 as potential elements of the ALT pathway like UBR5, NSMCE2, RFC4, MCPH1, RECQL4, LRATD2, RAD1, RAD21, NBN, FBH1, RAD54B, ATR, SMG7, and TERF1 [SENP5, NSMCE2, NBN, ATR, and TERF1 are involved in APBs formation [NSMCE2, RFC4, CCT5, NBN, and TERF1 to be expressed in the telomeres of ALT+ cell lines [Along with nd TERF1 . Likewisormation . Additioll lines .NSMCE2 recruitment is essential for APBs correct functioning [SMC5/SMC6 complex, in which inhibition is known to disrupt APBs formation [NBN is part of the MRN complex (MRE11/RAD50/NBN) that helps in the assemble of the G-overhang in the lead telomeric strand [ATM to the telomeres, allowing the invasion of adjacent telomeric DNA to be used as a template for telomere extension [Some of the genes proposed have been predicted to play an important role in ALT-related mechanisms; for instance: ctioning , it is aormation . NBN is c strand ; additioxtension .MCPH1 and ARID1A bind to the telomerase reverse transcriptase (hTERT) and regulate its expression [LRATD2, is known to be upregulated in cells with short telomeres [ATR is important in the assembly of the telomerase complex [TERF1 is overexpressed in cells with long telomeres [RECQL4 is associated with shelterin proteins POT1, TRF1 and TRF2 in the formation and stability of the t-loop and its knockdown causes telomere disfunction, possibly favoring the start of the ALT mechanism [Moreover, pression ; the oncelomeres . ATR is  complex . TERF1 ielomeres  and lastechanism ,65.NSMCE2, RFC4, NBN and ATR are recognized as enhancers, while, SENP5, UBR5, RAD21, MCPH1, RECQL4, ARID1A, CCT5, LRATD2, RAD1, FBH1, RAD54B, SMG7, and TERF1 are qualified as ambiguous. This highlights the importance of performing ex-vivo validations in the ALT-associated genes proposed in this study in order to improve the understanding of their role in the switching, progression or maintenance of ALT.According to the TelNet database, based on their predicted activity in the ALT pathway, TM genes are classified as enhancers, repressors or ambiguous  Figure . Only NSNADK, a kinase predicted to be a negative regulator of telomerase [In addition to the 20 genes proposed for ex-vivo studies, we classified the genes based on their representation in the different types of ALT tumors proposed in lomerase  or ERCC1lomerase .p < 0.001) were selected; 35% of the 103 proteins analyzed were involved in telomere organization, maintenance and telomeric DNA binding, while 22% are crucial for HR and HRR, which altogether with non-homologous end joining (NHEJ) have an important role in the mechanism by which ALT+ cells extend its telomeres [In order to understand the role of the proteins in ALT+ cells, a protein-protein interaction network was performed using STRING a. The moelomeres ,68.RAD52, both pathways relying on the activation of POLD2 and POLD3 [Break-induced replication (BIR) is a specialized form of HR . BIR is nd POLD3 ; howevernd POLD3 . Hence, nd POLD3 ,70, thisMoreover, we performed an enrichment analysis for 103 proteins using g:Profiler, which searches a collection of proteins with pathways, networks, GO, and cancer phenotypes . The GO RAD54B, NSMCE2, TERF1, SMG7, RECQL4, ATR, PRKDC, RAD1, RFC4, and NBN are directly involved in pathways like DDSB, HR, HDR, and HRR among others, which are ALT-related processes. The enrichment analysis concluded that genes related to HR are upregulated and amplified in the PCA samples studied, which, as stated before, could be triggering the expression of ALT-dependent pathways.Furthermore, we selected the proposed ALT-related genes in our study, the most significant pathways from the PPi and enrichment analysis and constructed a CIRCOS plot b to obseIn this study, we conducted a genomic analysis of 411 TM genes in 31 PCA studies and with the aid of different in-silico approaches, we proposed 20 candidate genes that are highly mutated as well as up and down regulated in different cancer types, which can be useful for future studies as potential molecular targets for the detection and understanding of the ALT pathway. Additionally, based on the prevalence of ALT+ tumors reported in this study, we strongly suggest to expand the study of ALT to a wide group of cancer types. Finally, we showed the need of a deep study of expression profiles and ex vivo assays to target TM genes that can help to understand the up and down regulation of ALT-related pathways and can provide guidance in the development of new therapies for the treatment of ALT+ tumors. We strongly believe this new decade will be promising for the study and understanding of this mechanism.


Despite their high prevalence, the pathophysiology of allergic rhinitis (AR) and chronic rhinosinusitis (CRS) remains unclear. Recently, transient receptor potential (TRP) cation channels emerged as important players in type 2 upper airway inflammatory disorders. In this review, we aim to discuss known and yet to be explored roles of TRP channels in the pathophysiology of AR and CRS with nasal polyps.TRP channels participate in a plethora of cellular functions and are expressed on T cells, mast cells, respiratory epithelial cells, and sensory neurons of the upper airways. In chronic upper airway inflammation, TRP vanilloid 1 is mostly studied in relation to nasal hyperreactivity. Several other TRP channels such as TRP vanilloid 4, TRP ankyrin 1, TRP melastatin channels, and TRP canonical channels also have important functions, rendering them potential targets for therapy.The role of TRP channels in type 2 inflammatory upper airway diseases is steadily being uncovered and increasingly recognized. Modulation of TRP channels may offer therapeutic perspectives. Rhinitis and rhinosinusitis represent two clinical entities of chronic upper airway diseases, characterized by inflammation of the (para)nasal mucosa that results in classic pathological features such as rhinorrhea, post-nasal drip, nasal obstruction, nasal itch, sneezing, loss of smell, and/or facial pain and pressure. Based on patient history and clinical findings, i.e., symptom severity, atopy, presence/absence of nasal polyps, and various comorbidities, different phenotypes of rhinitis and rhinosinusitis are defined. Additionally, the underlying mechanisms, or endotypes, are diverse and even overlapping, making it sometimes difficult to clearly distinct different phenotypes •.Considering rhinitis, three major phenotypes can be recognized and distinguished: allergic rhinitis (AR), infectious rhinitis, and the heterogenous group of non-allergic rhinitis (NAR) . In caseNasal congestion, edema, mucus production, and—in the case of CRSwNP—nasal polyp formation lead to nasal obstruction, whereas rhinorrhea and postnasal drip are the result of mucus production and often impaired mucociliary clearance –6. In ARIn Europe, up to 30% and over 10% of the general population suffers from AR  and CRS Both AR and CRSwNP share many similarities and are mainly driven by type 2 inflammation Figs.  and 2 11•. IndeeIn the last decades, transient receptor potential (TRP) cation channels emerged in the field of upper airway inflammation as active players in the context of NHR, which is present in the majority of patients with rhinitis, CRS, and asthma , 20. TRPTrp gene was discovered in 1989 [Since the first mammalian  in 1989 , TRP cha in 1989 , and bas in 1989 , 37. TRP in 1989 , 38 of f in 1989 . The sel in 1989 , 40. TRP in 1989 –45. Besi in 1989 , 46–50•.+ and Ca2+ concentrations and therefore membrane depolarization [TRP channel activation, i.e., the transition from a closed to open pore conformation, leads to cation entry at physiological resting membrane potentials, resulting in the increase of intracellular Narization . TRP charization .2) into diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3) [2 [2, generating arachidonic acid of which the metabolites, such as 12-hydroxyperoxyeicosatetraenoic acid or prostaglandin E2, can activate TRP channels [2+-permeable, they could play a role in the phenomenon of store-operated Ca2+ entry (SOCE) [2 into DAG and IP3, the latter binds on its receptor located on the endoplasmic reticulum, leading to release of Ca2+ from this intracellular store [2+-release activated channels (CRAC) formed by Orai1 subunits leading to a consistent Ca2+ influx and store-operated Ca2+ channels (SOC) formed by Orai1 and TRPC proteins resulting in non-selective cation influx [In the upper airways, TRPV1 and TRPA1 are studied the most. They have been reported to be expressed in T cells, mast cells, and afferent neurons and can be activated directly  or indirectly (via activation of G protein-coupled receptors or receptor tyrosine kinases) ••. Activte (IP3) ••, 52. T(IP3) [2 ••, 52. F(IP3) [2 ••, 52. Achannels ••, 52. Ly (SOCE) . After har store ••, 53. Nn influx , 53.2+ plays an important role in intracellular signaling and cellular responses in T cells [2+ concentration [2+ influx from the extracellular environment and intracellular stores. Subsequent activation of CRAC results in a sustained Ca2+ influx [+ T cells, there is evidence of expression of TRPA1, TRPC1/2/3/5, TRPV1/2/3/4, and TRPM1/2/4/5/6/7  channel, depolarizes the plasma membrane, hence reducing the driving force for SOCE-mediated Ca2+-entry, preventing intracellular Ca2+-overload [TCR-mediated activation induces release of adenosine 5′-diphosphoribose (ADPR) from the endoplasmic reticulum, which subsequently activates TRPM2 channels . Furtheroverload , 81.Lastly, as stated above, TRPC1 and TRPC3 are involved in SOCE, although their precise roles remain to be clarified , 82. In 2+ is involved in activation of transcription factors, as well as in the production and release of mast cell mediators [2+ mobilization into the cytoplasm from the extracellular space and from intracellular stores, followed by SOCE [2+ concentration, different cellular responses are triggered, such as degranulation and chemotaxis [2+ influx triggers the release of mast cell mediators such as histamine and eicosanoids to the extracellular environment [TRP channels expressed on mast cells include TRPC1–7, TRPV1/2/4/6, TRPM2/4/7/8, and TRPA1 . In thesediators , 42. The by SOCE . Dependiironment .In resting mast cells, TRPA1 is localized on intracellular vesicles, where it can interact with secretogranin III, and thus, it may play a role in formation of mast cell granules . Also, T2+ current, but this does not lead to mast cell degranulation [TRPV1 stimulation with its specific agonist capsaicin results in an inward Canulation . In contnulation , 66.2+-dependent mast cell migration [TRPM4 is rapidly activated after FcεRI stimulation, depolarizing mast cells and hence counter-acting SOCE, similar to the situation in T cells . TRPM4 sigration .2+-permeable SOC channels [Lastly, TRPC channels are activated after G protein-coupled receptor or receptor tyrosine kinase stimulation, but are also involved in SOCE , 82. Likchannels .TRP channels are expressed in epithelial cells of the nasal and paranasal mucosa, but their functional roles remain to be fully determined. Immunohistochemistry of the nasal mucosa showed expression of TRPV1–4 around seromucinous glands and capillary endothelial cells in the lamina propria, as well as in epithelial cells . In a stStimulation of the menthol- and cold-activated TRPM8 in sensory neurons induces a subjectively enhanced nasal patency without any objective ground , 92. ConIn trigeminal neurons, TRP channels are highly expressed in unmyelinated sensory C fibers and are suspected to play key roles in the phenomenon of NHR ••. TRP c2, which skews towards a type 2 inflammation and yields anti-inflammatory properties [Studies on TRP channels in upper airway inflammation have focused mainly on idiopathic rhinitis patients, where an overexpression of TRPV1 and increased levels of SP in nasal secretions were linked to neurogenic inflammation and NHR ••. Howevoperties , sensitioperties ••, 121 aoperties , 123. InAs for idiopathic rhinitis, up to two-thirds of AR and CRS patients report NHR , 124. StTrpv1 knockout mice, as well as mice treated with a pharmacological antagonist of TRPV1, exhibited less type 2 inflammatory mediators after allergic sensitization [Considering TRPV1 in AR, several observations are interesting. For instance, one study found TRPV1 expression based on RT-q-PCR to be significantly decreased in nasal mucosa of AR and NAR patients , whereastization . FurtherIn CRSwNP patients, one study found decreased TRPV1 and TRPA1 mRNA levels in nasal mucosa, whereas TRPV1 was upregulated in case of comorbid asthma or allergy . AnotherLastly, the activation of upper respiratory epithelial TRPV1 enhances the release of pro-inflammatory mediators , 73, 74 In summary, TRPV1 is mostly associated with NHR and neurogenic inflammation. However, it also facilitates T cell activation with consequent release of inflammatory mediators, playing a role very early in the pathophysiological pathway of type 2 inflammation. In addition, the activation of TRPV1 in upper respiratory epithelial cells could enhance the inflammatory process. Hence, TRPV1 is implicated in all aspects of the pathophysiological mechanisms underlying AR and CRSwNP and leading to nasal symptoms, rendering it a worthy candidate for pharmacological modulation.Despite the scarcity of studies on TRPA1 in specific inflammatory disorders of the upper airways, this TRP channel deserves to be mentioned. TRPA1 is expressed in sensory afferent neurons in both upper and lower airways and can be activated by a plethora of noxious exogenous stimuli , 140. FoSome studies in the lower airways found TRPA1 in immune cells and in airway epithelial cells . Here, aAs mentioned earlier, NHR is a key feature of rhinitis and of rhinosinusitis. NHR is currently best diagnosed with a cold, dry air provocation test , and TRP2+ concentration, which in turn triggers the release of nitric oxide and an increase in the ciliary beat frequency. Hence, it was postulated that TRPV4 plays a role in protective responses to gram-negative bacterial infections [TRPV4 is ubiquitously expressed and can be activated by a variety of both physical and chemical stimuli, including hypotonicity, high temperature, and metabolites of arachidonic acid . It medifections . This maTRPV4 is currently being investigated as a potential therapeutic target in cough arising from the lower airways, pulmonary edema, and other respiratory and non-respiratory diseases , 147. DuActivation of respiratory epithelial TRPM8, for example by menthol or by cooling, is suspected to lead to increased mucus production . This li2+ to enter the cell, possibly reducing pro-inflammatory activity of T cells [Given their role in both T cell and mast cell activation, the inhibition of TRPM2 , 68 and  T cells , 81, 64  T cells . TherefoTRPC6 is overexpressed on epithelial cells of CRSwNP patients, and its activation induces production of IL-1ß, IL-5, and IL-25 . TRPC5 i2+-dependent signaling pathways and contribute to a plethora of cellular processes. In the last decades, their cellular functions and their roles in multicellular processes and interactions have been studied intensively. In type 2 upper airway inflammatory disorders, the battlefield is mainly occupied by T cells, mast cells, epithelial cells, and sensory neurons, each releasing their respective mediators and hence interacting with each other, influenced by neuro-immune interactions and barrier defects. The role of TRP channels in these diseases is only starting to be clarified. Even though potential therapeutic targets have already emerged, a TRiP to successful treatment of type 2 upper airway inflammation awaits.TRP channels are widely expressed, conserved cation channels that regulate intracellular Ca


Diabetic kidney disease (DKD) is a microvascular complication of diabetes mellitus (DM) and comprises multifactorial pathophysiologic mechanisms. Despite current treatment, around 30-40% of individuals with type 1 and type 2 DM (DM1 and DM2) have progressive DKD, which is the most common cause of end-stage chronic kidney disease worldwide. Mesenchymal stem cell- (MSC-) based therapy has important biological and therapeutic implications for curtailing DKD progression. As a chronic disease, DM may impair MSC microenvironment, but there is compelling evidence that MSC derived from DM1 individuals maintain their cardinal properties, such as potency, secretion of trophic factors, and modulation of immune cells, so that both autologous and allogeneic MSCs are safe and effective. Conversely, MSCs derived from DM2 individuals are usually dysfunctional, exhibiting higher rates of senescence and apoptosis and a decrease in clonogenicity, proliferation, and angiogenesis potential. Therefore, more studies in humans are needed to reach a conclusion if autologous MSCs from DM2 individuals are effective for treatment of DM-related complications. Importantly, the bench to bedside pathway has been constructed in the last decade for assessing the therapeutic potential of MSCs in the DM setting. Laboratory research set the basis for establishing further translation research including preclinical development and proof of concept in model systems. Phase I clinical trials have evaluated the safety profile of MSC-based therapy in humans, and phase II clinical trials  still need to answer important questions for treating DKD, yet metabolic control has already been documented. Therefore, randomized and controlled trials considering the source, optimal cell number, and route of delivery in DM patients are further required to advance MSC-based therapy. Future directions include strategies to reduce MSC heterogeneity, standardized protocols for isolation and expansion of those cells, and the development of well-designed large-scale trials to show significant efficacy during a long follow-up, mainly in individuals with DKD. The global diabetes mellitus (DM) prevalence in 2019 was estimated at 9.3% (463 million) in adults aged 20-79 years, rising to 10.2% (578 million) by 2030 and 10.9% (700 million) by 2045 [Using the WHO  database, the International Diabetes Federation documented that 8.4% of all-cause deaths were attributable to DM in adults aged 20–79 years, almost 5.1 million deaths [Diabetic kidney disease (DKD) is a microvascular complication of DM and the most common cause of end-stage kidney disease (ESKD) worldwide, with approximately 30% of patients with type 1 DM (DM1) and approximately 40% of patients with type 2 DM (DM2) developing DKD, as reviewed elsewhere [2, (ii) stage 2 (incipient nephropathy): microalbuminuria (30-299 g/g Cr) and eGFR ≥ 30 ml/min/1.73 m2, (iii) stage 3 (overt proteinuria): macroalbuminuria (≥300 g/g Cr) or persistent proteinuria (≥0.5) and eGFR ≥ 30 ml/min/1.73 m2, (iv) stage 4 (kidney failure): any albuminuria status and eGFR < 30 ml/min/1.73 m2, and (v) stage 5 : any status on continued dialysis therapy [Natural history of DKD comprises hyperfiltration, progressive albuminuria, decrease in eGFR (estimated glomerular filtration rate), and, ultimately, ESRD. Yet, albuminuria is a continuum; eGFR deterioration can start to decline before progression to overt nephropathy, which can be explained by other risk factors, such as obesity, hypertriglyceridaemia, hypertension, and glomerular hyperfiltration . Thus, a therapy .Histologically, metabolic changes associated with DM lead to glomerular hypertrophy, glomerulosclerosis, arteriolar hyalinosis, arteriosclerosis, tubule-interstitial inflammation, and fibrosis. The main glomerular changes consist of thickening of the glomerular basement membrane (GBM), expansion of the mesangial matrix, atrophy and loss of podocyte pedicels associated with effacement, and diffuse or nodular intercapillary glomerulosclerosis (Kimmelstiel-Wilson lesion) .β-cell dysfunction) and haemodynamic changes (systemic hypertension) characterizes DKD pathophysiology. In addition, DKD is associated with endothelial dysfunction; activation of RAAS ; increase in AGEs (advanced glycation end products); elevation of NADPH oxidase; upregulation of GLUT1; generation of reactive oxygen species (ROS); upregulation of growth factors, such as VEGF  and TGF-β (transforming growth factor-β); activation of aldose reductase and the polyol pathways; mitochondrial dysfunction; downregulation of adiponectin; and nitric oxide (NO) loss, as reviewed elsewhere [κB; and inflammation, which is associated with inflammatory interleukins (IL), cytokines, and chemokines , CSF-1 (colony stimulating factor-1), MCP-1 (monocyte chemoattractant protein-1), and MIF (macrophage inflammatory factor)). Exacerbated production of profibrotic cytokines (CTGF (connective tissue growth factor) and TGF-β) associated with fibrosis is also involved in DKD. Collectively, all those mechanisms contribute to DKD progression and to both functional (declining eGFR and proteinuria) and structural  kidney damage, which lead ultimately to systemic complications .Systemic inflammatory milieu due to metabolic dysregulation  prevention of obesity, (2) screening for and prevention of diabetes in an at-risk population, (3) glycemic control once diabetes develops, (4) blood pressure (BP) control once hypertension develops, (5) screening for diabetic chronic kidney disease (CKD), (6) RAAS inhibition/blockade in those with diabetic CKD, and (7) control of other cardiovascular (CV) risk factors such as management of low-density lipoprotein cholesterol (LDL-C) , 10.Despite diabetic patients being treated with angiotensin-receptor-blockers (ARBs), renal disease progression risk over 2 years increases with increasing proteinuria and albuminuria and decreasing eGFR . To noteNovel drugs have been recently associated with clinical benefit profiles, which should be considered in the decision-making process when treating patients with DM2. Glucagon-like peptide 1 receptor agonists (GLP1-RA) and sodium-glucose cotransporter-2 inhibitors (SGLT2i) reduce atherosclerotic major adverse cardiovascular (CVs) events to a similar degree in patients with established atherosclerotic CV disease, whereas SGLT2i have a more marked effect on preventing hospitalization for heart failure and progression of DKD , 15.in vitro and in vivo studies using syngeneic, autologous, allogeneic, or xenogeneic mesenchymal stem cells (MSCs) for treating DKD. We will describe the main findings of MSC-based therapy in preclinical and clinical studies and discuss the benefits, outcomes, and challenges of that therapy for halting DKD progression.In the DKD treatment setting with drugs and lifestyle changes, novel approaches are further required to halt the progression of DKD or regenerate the damaged tissue, such as cell therapy . In thisMSCs, commonly referred to as mesenchymal stem cells or mesenchymal stromal cells, are a diverse population of cells with a wide range of potential therapeutic applications for different organs and tissues. MSCs can be obtained from many tissue sources, consistent with their broad, possibly ubiquitous distribution.Historically, MSCs were isolated from bone marrow (BM-MSC) and spleen from guinea pigs by Friedenstein et al. . They obBM-MSCs can be isolated by (a) using gradient centrifugation (Ficoll or Percoll) to separate nonnucleated red blood cells from nucleated cells, (b) taking advantage of their ability to adhere to plastic, (c) taking advantage of the ability of monocytes to be separated from BM-MSCs by trypsinization .st century, in vivo studies documented that human BM-MSCs differentiated into endodermal-derived cells [in vivo and therefore is not the main mechanism of tissue repair and regeneration.During the 1980s, BM-MSCs were found to be able to differentiate into osteoblasts, chondrocytes, adipocytes, and muscle tissue , 21. Dured cells , 23, cared cells , and rened cells , 26. HowMore recently, BM-MSC secretome has demonstrated potential clinical applications and includes both soluble proteins  and factors released in extracellular vesicles, for example, microvesicles (size 100-1000 nm) and exosomes  . These ein vitro from different organs and tissues  [5‐1 × 106 AT-MSCs. This is compared to 5 × 102‐5 × 104 of BM-MSCs isolated from 1.0 g of BM aspirate [Of importance, MSCs possess ubiquitous distribution in perivascular niches and can be derived and propagated na cava) , 32. Mosaspirate .Isolation of MSCs from AT is based on mincing fat tissue, followed by several washings in order to remove contaminating hematopoietic cells, incubation of tissue fragments with collagenase, and centrifugation of the digest, thereby separating the floating population of mature adipocytes from the pelleted stromal vascular fraction . UCB-MSCMSC populations originating from different tissues and organs exhibit similar morphology and, to a certain extent, surface marker profile . On the in vitro [The International Society for Cell Therapy (ISCT) established the characteristics of MSCs from all sources, either autologous or allogeneic: (1) adherence to plastic under standard culture conditions; (2) expression of CD73, CD90, and CD105 surface molecules in the absence of CD34, CD45, HLA-DR, CD14 or CD11b, CD79, or CD19 surface molecules, as assessed by flow cytometry analysis; and (3) differentiation capacity for osteoblasts, adipocytes, and chondroblasts in vitro . In compin vitro .in vitro and in vivo data; and (iii) a robust matrix of functional assays to demonstrate the properties of these cells associated with the intended therapeutic mode of actions. In addition, basic assays for MSC-based products comprise donor screening, viability test, purity test , safety test , identity tests (immunophenotypic profiles), and potency tests . All of these procedures should be done in a Good Manufacturing Practice (GMP) facility.A recent update from ISCT includes analyses that mitigate the heterogeneity of MSCs, such as assays that demonstrate the secretion of trophic factors, the modulation of immune cells, and other relevant functional properties, such as angiogenesis . The ISCγ (IFN-γ primed MSCs) and subsequent analysis of various ribonucleic acids  and (b) coculture of MSCs with human peripheral blood mononuclear cells (PBMCs) and analysis of the signature of the secretome in relation to cytokine/chemokine secretion and T cell proliferation [To assess MSC self-renewal capacity, doubling time and CFU-f are broadly used. In a particular DM setting, immunological assays can be based on activation protocols that discriminate between TLR- (Toll-like receptor-) 4-dependent phenotype MSC-1 and TLR3-dependent MSC-2 phenotype . That poferation . Cocultuin vitro DKD milieu is challenging since cell-cell and cell-matrix interaction is severely affected during disease progression. The most frequent approach is to evaluate the cells under normal glucose medium (5.5 mmol/l) and high glucose (25-30 mmol/l or less frequent 40 mmol/l). Mannitol (20 mmol/l) associated with normal glucose (5.5 mmol/l) is used as a control of osmolality. Peroxide hydrogen and TNF-α may be added to the medium as inducers of oxidative stress [in vitro , the amount of cells , the type of cell interaction , and duration of the coculture  were broadly tested in the literature.To recapitulate, the e stress  and infle stress , respectImmortalized mouse podocytes cultured in high glucose medium and cocultured directly with BM-MSC transfected with miR124a, for 24 h, exhibited increased viability and decreased apoptosis (decrease in caspase-3 and Bax gene expression and increase in Bcl2 gene expression) . Mouse pβ and phosphorylated SMAD2/3 proteins, which were abrogated by BMP-7 antibody [5 cells/well) in a Transwell® chamber for 72 h led to an increase in lipoxin A4, a key lipid involved in inflammation resolution [For glomerular mesangial cells (GMCs) cultured in high glucose medium, direct coculture with BM-MSC (ratio 10 : 1) or MSC-conditioned medium for 72 h decreased equally TGF-antibody . Likewissolution .5 cells/well) using a Transwell® chamber inhibited apoptosis of those cells, induced klotho expression, and downregulated the Wnt/β-catenin signaling pathway [α may also mimic the DKD microenvironment [α. UCB-MSC increased cell viability, ATP production, and E-cadherin expression, as opposed to a decrease in fibronectin, SGLT2, pNF-κB p65, and MCP-1.For renal tubular epithelial cells (TECs) cultured in high glucose medium, the coculture for 24 h with AT-MSC (1 × 10 pathway . In addiironment . In thatβ1) [Not only MSCs but also EXOs cocultured for 96 h with TECs in primary renal cell culture of streptozotocin- (STZ-) induced diabetic rats entailed in antiapoptotic and antidegenerative effects (increase in ZO-1 and lectin expression and decrease in TGF-β1) .2O2 conditioning, which was abrogated by MSC treatment and activation of the β-catenin-dependent Wnt signaling pathway [Endothelium may also be damaged during DM progression. Thus, the murine islet microvascular endothelium cell line experienced apoptosis and endothelial cell activation (increase in VCAM (vascular cell adhesion molecule) expression and reduction in eNOS  phosphorylation) upon H pathway .4 cells/well) with LPS-treated macrophages , at a 1 : 5 ratio for 6 h, led to a decrease in IL-1β, IL-6, MCP-1, and TNF-α expression [β, TNF-α, IL-6, and iNOS (inducible nitric oxide synthase)), which was mediated by the increase in arginase 1 production [Therapeutic potential of MSCs can also be verified in a coculture platform with other cells that play a role in DKD progression, such as macrophages. Indirect coculture of BM-MSC ; higher levels of IGF-1 (insulin growth factor-1), HGF (hepatocyte growth factor), and VEGF; and upregulation of β-catenin signaling when compared to renal-derived Sca-1+ MSC from wild-type mice [Aging has also an adverse impact on MSC function and possesses biological and therapeutic implications. Moreover, CKD and DM are linked to accelerated aging. The p66 protein is related to aging and controls cellular response to oxidative stress, senescence, and apoptosis. Renal-derived Sca-1ype mice .4‐3.0 × 106), the beneficial outcomes for kidney recovery favored MSC treatment [MSC-based therapy is a promising strategy for accelerating kidney recovery, repairing and regenerating tissue damage after acute injury following ischemia-reperfusion, kidney transplant, and drug-mediated toxicity, as reviewed elsewhere . In a mereatment .Of importance, MSC efficacy is challenged by several factors, such as viability, cell source, MSC phenotype, homing capacity, route of delivery, site of infusion, number of infusions, cell passage, cell potency, severity of condition, and target impact . In the Next, we discuss some key aspects of MSC-based cell therapy in preclinical studies.γ (interferon-γ) and TNF-α, MSCs acquire an immunosuppressive phenotype (MSC2) and through Toll-like receptor- (TLR-) 3 lead to an increase in production of TGF-β, IDO , NO (nitric oxide), and PGE2 (prostaglandin E2). These events stimulate the amount of CD4+CD25+FoxP3+ T regulatory cells. Conversely, in the absence of an inflammatory environment (low levels of IFN-γ and TNF-α), MSCs acquire a proinflammatory phenotype, and through TLR4, LPS (lipopolysaccharide), and high levels of chemokine C-X-C motif ligand (CXCL)9, CXCL10, MIP- (macrophage inflammatory protein-) 1α, MIP-1β, and CCL5/RANTES , but low levels of IDO, NO, and PGE2, activation of cytotoxic T lymphocytes is triggered.Emerging concepts indicate that MSCs may function as sensors and switchers of inflammation, which may explain their immunomodulatory properties , 59. In γ and TNF-α).Interaction of MSCs and monocytes play also a key role in our understanding of mechanisms of MSC-mediated tissue regeneration , 59. Whe+ T lymphocytes and donor-specific CD8+ T lymphocyte cytolytic response [However, further investigation is warranted to verify whether MSC phenotype changes in accordance with DKD progression. In other settings, such as kidney transplant, MSC infusion posttransplant allowed their preferential recruitment in the inflammatory milieu of the graft created by ischemia/reperfusion injury, and once in that environment, MSC contributed to upregulation of inflammation, thereby causing premature graft dysfunction . By contresponse  and mighresponse .μm [Stem cell route delivery  may affect MSC efficiency for kidney repair and regeneration in different models of acute and chronic kidney injury. The intravenous route is the route used most often, to inject not only MSCs –67 but aμm , 71. Simμm , 73. SodHowever, infused human MSCs are able to migrate beyond the lungs after intravenous administration in a rodent model of cisplatin-induced acute kidney injury and may be detected in peritubular areas, where they ameliorated renal cell apoptosis and increased cell proliferation .Intra-arterial routes for delivering progenitor/stem cells include intracarotid , intracaAlthough intraparenchymal  administration of progenitor/stem cells or MSCs has beneficial effect on kidney repair , 85–89, Stromal-derived factor-1 (SDF-1), also known as CXCL12, and its receptor C-X-C chemokine receptor 4 (CXCR4) axis is a crucial key pathway in cell trafficking.After acute kidney injury, the levels of SDF-1 mRNA levels increase more than 2.5-fold and remain high as ~2.0-fold after 24 h within kidney cortex tissue . That inThere are several animal models of DKD in rodents, which mimics DM in humans either DM1 or DM2. Therefore, DM and subsequent DKD can be obtained by genetic manipulation, induced by drugs (streptozotocin or STZ) or high-fat diet, or even a combination of approaches, including uninephrectomy to accelerate DKD progression, as reviewed elsewhere .Thus, pharmacologic induction of DKD with STZ, with or without accelerating factors, such as high-fat diet, uninephrectomy, or use of the nonobese diabetic (NOD) strain, has been the most common rodent model of DKD to study the potential therapy of MSCs .The Animal Models of Diabetic Complications Consortium (AMDCC) defined the following criteria for validating a progressive mouse model of DKD : (i) greob/ob; knockout for leptin) that reflect human DKD [ob/ob models develop proteinuria in a time-dependent manner, mesangial expansion, thickening of GBM, widening of podocyte foot process, podocyte apoptosis, glomerular sclerosis, and reduction of podocyte genes and protein. Notably, BTBRob/ob mice comprise a reversible model of DM upon leptin administration [Recent models of DM1  and DM2  [1 (cholecystokinin) receptors represents a broadly established model of DM2, which develops proliferation of the mesangial matrix, GBM thickening, diffuse glomerulosclerosis, nodular lesions, tubular atrophy associated with mononuclear cell infiltration, and fibrosis [NOD mice develop autoimmune insulitis caused by polygenes including specific MHC class II alleles and many non-MHC loci, mimicking DM1 . NOD mic (db/db) . They defibrosis . Other rfibrosis .In Briefly, these studies provided evidences that MSC-based therapy may decrease fasting blood glucose (FBG) and glycated haemoglobin (HbA1c) in either DM1 or DM2 animals, and in DM1 animals, plasmatic insulin levels increased or exogenous insulin requirement decreased. Likewise, MSC-based therapy has important therapeutic implications in the DKD setting, providing insights into cellular and molecular mechanisms. Therefore, MSCs contributed to improving functional parameters, such as the increase in glomerular filtration and the decrease in albuminuria and structural parameters. The studies indicated an improvement in renal histology and the curtailing of biological processes of inflammation, cell death (apoptosis and necrosis), oxidative stress, and fibrosis. In addition, MSC-based therapy promoted preservation of renal mass, upregulation of tubular epithelial and podocyte genes, augmentation in growth factors within the kidneys, decreasing endothelium damage, amelioration of tubular glucotoxicity by decreasing cellular glucose uptake in the kidneys, and increasing the antiaging klotho protein.β-cells may also represent a promising strategy to treat DM and clinical complications, as documented by the amelioration of endothelium activation by decreasing fibrinogen levels, blood pressure, cytoplasmic calcium, and apoptosis (p53 and Bax), as well as by improving cardiac parameters in STZ-induced diabetic rats [Differentiated BM-MSCs to insulin-secreting tic rats .β binding protein), and LTBP-2, as well as by promoting vascular formation in vivo [α) and reduced antiangiogenic genes . Extracellular matrix-related proteins  were also higher in diabetic secretome. These data indicate that BM-MSCs from DM2 rats have a unique secretome with distinct angiogenic properties and provide new insights into the role of BM-MSCs in aberrant angiogenesis in the diabetic milieu.Likewise, secretome of BM-MSC obtained from Zucker DM2 fatty rats improved endothelial cell function by increasing ~3-fold the formation of tubule-like structures and migration of these cells, which was mediated by IGF-1, LTBP-1 (latent TGF- in vivo . In addiin vitro) and self-renewal, which may compromise the efficiency of direct self-repair and autologous cell therapy [in vivo [The hyperglycemic milieu may also adversely impact MSC functionality. Therefore, AT-MSC extracted from Zucker diabetic fatty rats exhibited downregulation of markers of pluripotency , TNF-α, as well as alpha muscle actin, which represents the stress fiber, and XBP-1, which represents endoplasmic reticulum stress), greater senescence, lower viability and homing capacity, increased apoptosis, and a reduction in clonogenic and multidifferentiation potentials [Likewise, MSCs extracted from rodents with DM2 or large animals with metabolic syndrome have morphological abnormalities (larger number of degenerated mitochondria and marked expansion of endoplasmic reticulum), less proliferative potential associated with an increase in doubling time, alteration in gene expression , such aberrations were observed after several passages in vitro , as wellnjection  or promonjection . The injnjection .in vitro, and injection back into the patient, thus avoiding complications resulting from graft rejection and/or the need for an immunosuppressive regimen. Therefore, while patient-derived (autologous) MSC may be the safer choice in terms of avoiding unwanted immune response, factors including donor comorbidities  and aging may preclude those cells from use.BM-MSCs are the main source of autologous cell transplantation for various diseases including DM-related micro- and macrovascular complications . Therapyin vitro studies documented the differentiation potential of human AT-MSCs into islet-like cells when these cells were obtained from healthy individuals who underwent abdominoplasty or liposuction [Notwithstanding recent promising results with MSC therapy in several diseases, moving the concept forward toward the DKD setting should be critically assessed by looking for intrinsic MSC abnormalities caused by the hyperglycemic milieu, which may adversely affect their therapeutic potential in diabetic patients. Thus, AT-MSCs extracted from diabetic individuals have a greater capacity for adipogenic differentiation, but less chondrogenic and osteogenic differentiation , 131. Coosuction –135. TraNot only the source of MSCs but also the type of DM may affect the therapeutic potential of MSCs. MSCs extracted from DM1 individuals exhibited preserved morphology, growth kinetics, multipotency, and proliferative, immunomodulatory, immunosuppressive, and migratory capacities , 136.In contrast, MSCs extracted from individuals with DM2 have greater senescence, lower viability, increased apoptosis , less proliferative potential associated with increased doubling time, and a reduction in angiogenic potential , 137.CD105 (endoglin) is associated with angiogenesis , and itsIn line with the derangement observed in MSC-induced angiogenesis of rodents, AT-MSCs extracted from DM2 individuals with critical limb ischemia are dysfunctional, e.g., they exhibited a reduction in fibrinolytic activity and an increase in prothrombotic activity and PAI- levels. Those cells also possess lower efficiency of proliferation, migration, and CFU-f assay, as well as derangement in the PDGF (platelet-derived growth factor) signaling pathway . PDGF siin vitro [The change in the secretome of diabetic MSCs grown even under normoglycemic conditions is related to the development of metabolic memory, a process in which hypomethylation in gene promoters leads to dysregulation of gene expression and implies the persistence of DM-related complications even when glucose returns to normal levels. That effect is supported by studies that show changes in glucose metabolism in diabetic MSCs and by the fact that their functional capacities were not altered by normalization of glucose levels in vitro , 122.in vitro rate, and HbA1c levels may play a role in that effect, indicating that higher rates of proliferation occur when HbA1c levels were 8-10% (versus HbA1c < 6.5%), yet serum derived from individuals with HbA1c > 10% exhibited a decrease in MSC proliferation [6/kg), exhibited a safety profile and contributed to decreasing proteinuria, yet not modifying eGFR in six patients [Therefore, serum obtained from DM2 individuals may increase the BM-MSC proliferation feration . On the patients . Other ppatients .in vitro immunosuppressive capacity when compared to normal individuals [Notably, BM-MSC of newly diagnosed (<6 weeks) DM1 individuals  presented similar morphology, immunophenotype, differentiation potential, gene expression of immunomodulatory molecules, and ividuals . Howevern = 10; mean age 22 years, range 18-35 years; 9 males), late stage of DM1 with severe renal failure , and healthy BM donors  [γ and TNF-α .In line with these findings, Davies et al. compared BM-MSC from individuals with newly diagnosed (<6 weeks) DM1  . They foob/J mice significantly reduced the excessive TNF-α response observed in diabetic mice and improved IL-10 secretion [α (HIF-1α), which led to an upregulation of angiogenic factors (VEGF and angiopoietin-1), neuroprotective factors , and cytokines with anti-inflammatory activity (IL-4 and IL-5) [in vitro, which was associated with a decrease in neuron death. PDGF pretreatment of human AT-MSCs extracted from DM2 individuals rescued these cells from the diabetic phenotype by improving the proliferation, migration, and the capacity of clot lysing and repairing skin wound in an animal model [To further substantiate the benefits of autologous MSC-based therapy, preconditioning strategies are key aspects to preserve MSC function, such as hypoxia culture, as previously described . In addiecretion . Iron chnd IL-5) . Deferoxal model .Another approach to decrease abnormalities of BM-MSC obtained from DM1 and DM2 animals is the coculture with human umbilical cord extracts (Wharton's jelly extract supernatant). Therefore, Wharton's jelly extract supernatant represents a cocktail of growth factors ; components of extracellular matrixes , L-glutamate, and EXOs may also ameliorate proliferative capacity, motility, mitochondrial degeneration, endoplasmic reticular functions, and EXO secretion in both DM1- and DM2-derived BM-MSC, since that supernatant provide the physiological environment to preserve MSC properties and functionality . These fIn conclusion, despite the fact that autologous MSC-based therapy has already been reported to ameliorate kidney injury, many difficulties must be overcome to successfully implement that therapy for treating DKD. Key aspects include the type of diabetes, time elapsed since the diagnosis due to cellular metabolic memory, and cell source, which may impair MSC functional properties.in vitro [In addition, some points are beyond the fact of choosing autologous or allogeneic MSCs for treating individuals with DM1 and DM2. Due to the expressive quantity of MSCs required to form a biobank and provide them to immediately infuse into patients, MSC expansion is a key aspect of cell therapy preparation. Both autologous and allogeneic MSCs cultured for a prolonged period may be affected by disturbance in the cellular structure and function. Chromosomal instability and aberrations have been shown in AT-MSCs after prolonged time in vitro , which lin vitro , 149. Liin vitro .Another important aspect that must be taken into account in cell therapy with MSCs is the fact that their beneficial effect can be neglected by the occurrence of adipogenic differentiation during long-term follow-up, which can contribute to glomerulosclerosis .The malignant transformation of MSCs has not been described in clinical trials , 152. AsClinical Trials web portal  with the keywords “mesenchymal stem cell” or “mesenchymal stromal cell” and “diabetes”. We defined inclusion criteria as completed studies that have reported results on PubMed. These studies were mainly single-center prospective phase I/II clinical trials, which evaluated safety and tolerability and explored the therapeutic effects of MSCs on beta-cell regeneration and the impact on fasting plasma glucose (FBG), HbA1c, endogenous insulin, and C-peptide increment and the reduction of daily insulin requirement ≥ 50%, which reached the efficacy level. A dose-escalating  randomized-controlled trial assessing one intravenous infusion of MPCs (rexlemestrocel-L) in DM2 individuals without DKD documented safety and efficacy of cell therapy [We have consulted the  therapy . In pati6 or 300 × 106, single intravenous dose), multicenter, and placebo-controlled trial, safety and efficacy of adult allogeneic BM-derived MPCs  were evaluated in type 2 diabetic individuals with DKD (eGFR 20-50 ml/min/1.73 m2). In terms of safety, no patients exhibit treatment-related severe adverse events and only one patient developed antibody specific to the donor HLA (antibody specificity to donor antigen (class I) B40; mean fluorescence intensity 530) at week 4 that were undetectable at week 12. The primary exploratory efficacy parameter comprised eGFR, so that the placebo-adjusted least square mean change in eGFR at week 12 was 4.4 ± 2.2 (p = 0.05) and 1.6 ± 2.2 ml/min/1.73 m2 (p = 0.47) for the 150 × 106 and 300 × 106 groups, respectively. Relative to placebo, there was a suggestion of stabilization of eGFR in the rexlemestrocel-L 150 × 106 group, most notably at the 12-week primary endpoint. Importantly, when subgroup analyses were performed (GFR ≤ 30 or >30 ml/min/1.73 m2), the subgroup with eGFR > 30 ml/min/1.73 m2 treated with 150 × 106 cells manifested a lower decrease in eGFR when compared to the control group at 12 weeks (p = 0.04). In addition, there was a statistically significant decrease in the median IL-6 values for the 300 × 106 group compared to placebo at week 12, but not for other markers .To note, there was only one multicentric study, which also included individuals with DKD . In thatin vitro and express genes responsible for insulin secretion .We observed a balanced distribution between allogeneic-MSCs and autologous-MSC-based studies for both DM1 and DM2 individuals. MSCs have also shown beneficial effects on glycemic control when combined to hematopoietic stem cells (HSCs) or BM-MNCs. However, studies have still not been able to establish insulin-free status in this group of patients, even by differentiation of human AT-MSC into insulin-secreting MSCs (AT-ISC-MSC) –158. Thaβ-cells with high-dose immunosuppressive drugs. A similar approach was also attempted to reset the deleterious immunologic system with a reconstituted one originated from autologous hematopoietic stem cells [β-cell mass and facilitate endogenous mechanisms of β-cell regeneration. For example, a nonmyeloablative low-intensity conditioning regimen combined to autologous AT-IS-MSC and HSC was based on rabbit antithymocyte globulin, methylprednisolone, and bortezomib [β-cell function from those studies should also be analyzed in light of the use of the immunosuppressive regimen per se.Likewise, a nonmyeloablative low-intensity conditioning regimen combined to MSC therapy failed to demonstrate insulin independence –158. Theem cells . The ratrtezomib . In addiMSC-based therapy was considered a safe procedure in all studies that verified the therapeutic potential of these cells. In a systematic review and meta-analysis of clinical trials that evaluated MSC safety in more than a thousand individuals diagnosed with other clinical conditions, a significant association between MSC infusion and fever was shown . HoweverIn terms of efficacy, both autologous- , 161–164Importantly, MSC differentiation, when exposed to a proinflammatory microenvironment, may result not only in upregulation of cell surface immunogenic molecules but also in a decrease in immunoregulatory or immunosuppressive molecule secretion, such as PGE2, as reviewed elsewhere . On the In addition, those studies also raised important questions regarding the protocols related to MSC source and viability, the number of infusions, the number of infused cells, routes of administration, the ability of MSCs to migrate to the injury site, the potency of the MSCs in the context of disease, model, and outcome measure . A key an = 22 studies), it was documented that almost 60% of DM1 individuals  became insulin-independent for a mean period of 16 months after HSC-MSC treatment (mean dose of 6.99 ± 3.28 × 106 cells/kg CD34+), as opposed to a negative response when these patients were treated with UCB cells  [6/kg to 1.88 × 107/kg; mean dose 2.6 ± 1.2 × 107) therapy was superior to BM-MSC (mean dose of 2.75 × 106/kg) therapy for DM1 individuals, when C-peptide levels were compared, but not in terms of decreasing HbA1c. For DM2 individuals , no conclusive recommendation was defined, yet DM-MNCs provided a better outcome when compared to UCB-MSCs in improving C-peptide levels and decreasing HbA1c. The administration of cell therapy early after DM diagnosis was more effective than intervention at later stages (relative risk = 2.0). To note, UCB cells (n = 3) and BM-MNCs (n = 107) were injected intrapancreatically, whereas UCB-MSCs (n = 22) were injected intravenously. In addition, mean doses of BM-MNCs were 17.29 × 108 cells/kg (mean number of CD34+ cells was 3.15 × 106) and mean doses of UCB cells were 5.29 × 109 (mean number of CD34+ was 2.88 × 106), whereas mean doses of UCB-MSCs were 1 × 106/kg (either intravenous or intrapancreatic). Whether the number of injected cells and the route of injection (intrapancreatic versus intravenous) are associated with better outcomes, further studies are warranted to reach a definitive conclusion. Importantly, the patient clinical condition may also play a role in cell therapy, as diabetic ketoacidosis may impair its efficacy.In a meta-analysis of UCB cell-, UCB-MSC-, BM-MSC-, and HSC-based therapies for both DM1 and DM2 individuals (6 × 106) . Likewisn = 6 studies, 206 patients), treatment with autologous BM-MNCs (dose ranged from 382.6 ± 107 to 2.8 ± 1.9 × 109 cells) was effective in reducing HbA1c by 1.18% and insulin requirement during a follow-up of 12 months [In another recent meta-analysis including DM2 individuals . Thus, the cultivation or preconditioning of MSCs in a hypoxia environment with 2% or 5% oxygen allows these cells to remain multipotent and have greater proliferative and migratory capacity, in addition to lower senescence rates , 179. Im in vivo .γ, TNF-α, PGE2, and NO oxide mitigated the heterogeneous behavior of MSCs on T lymphocyte proliferation assays and on late type hypersensitivity response [In order to reduce the heterogeneity of the MSC profile, which is defined by the different isolation and culture protocols, the preconditioning of these cells with proinflammatory factors has been the focus of investigation. Thus, the preconditioning of MSCs through stimulation with IFN-response .MSCs can also be tested as carriers of genes or genetic modifications. Due to their ability to migrate to injury sites, MSCs represent a robust platform for delivery of genes associated with regeneration and repair of renal tissue, functioning as a “Trojan Horse” . Thus, sα blocker [In addition, genetic modifications of MSCs, which are also very promising in the context of DKD, include the overexpression of erythropoietin, CXCR4, CTLA4Ig, and IL-10/selectin, as well as the transfection of minicircles containing biological drugs, such as etanercept, which is a TNF- blocker , and tra blocker .Despite MSC-based preconditioning treatment that has not been associated with harmful effects, further studies are required to verify its effectiveness in maintaining MSC properties.To advance MSC-based therapy, production of a large amount of these cells is challenging. Automated hollow-fiber bioreactors were validated to the development of large-scale manufacturing MSCs, providing cells with preserved characteristics and functionality when compared to the manual multilayer flask method , 185. ThMSC-derived secretome is a cell-free alternative for treating DM1 and DM2 individuals, which can bypass some issues related to autologous and allogeneic MSCs . Some adLaboratory research set the basis for establishing further translation research including preclinical development and proof of concept in model systems. Thus, animal models indicate that syngeneic, autologous, allogeneic, and xenogeneic MSCs are effective for treating metabolic dysfunction in the DM setting and for halting the progression of DKD. MSCs demonstrated efficacy in controlling several biological processes, such as apoptosis, autophagy, fibrosis, inflammation, and oxidative stress, as well as in ameliorating renal functional and structural parameters.BM-MSCs have been applied as the most valuable source of autologous cell transplants for diabetic complications in animals and humans. Despite differential gene expression, expanded MSCs from DM1 donors are phenotypically and functionally similar to healthy control MSCs with regard to their immunomodulatory, migratory potential, multilineage differentiation, and secretion of growth factors. MSCs from DM2 donors exhibit dysfunctional properties, such as senescence, angiogenesis impairment, higher rates of apoptosis, and lower clonogenic potential. Therefore, hyperglycemia may cause abnormalities in intrinsic BM-MSC, which might lose sufficient therapeutic effects in DM2 individuals. Of importance, DM2 donors are usually older and exhibit hyperglycemia for a longer duration, so that aging may play an additional role in MSC dysfunction. Therefore, preconditioning strategies can be used to recover the characteristics and functions of MSCs of diabetic patients before infusion and, thus, improve their performance in autologous therapies in terms of tissue repair and regeneration.Together, the bench to bedside pathway has been constructed in the last decade. Laboratory research set the basis for establishing further translation research including preclinical development and proof of concept in model systems . In FiguClinical trials using MSC-based therapy indicate that infusion of autologous or allogeneic MSCs is generally well tolerated. Phase II clinical trials including a longer period of observation will support the efficacy of MSCs. However, the use of these cells to treat diabetic individuals with DKD awaits clinical validation. In conclusion, MSC-based preclinical and phase I/II clinical data encourage the design of future large-scale controlled clinical trials that evaluate DKD response to MSC therapy, while rigorous reporting of safety and efficacy is still needed.


What are the risk factors associated with hospitalization, mechanical ventilation, and death among patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection?In this national cohort study of 88 747 veterans tested for SARS-CoV-2, hospitalization, mechanical ventilation, and mortality were significantly higher in patients with positive SARS-CoV-2 test results than among those with negative test results. Significant risk factors for mortality included older age, high regional coronavirus disease 2019 burden, higher Charlson Comorbidity Index score, fever, dyspnea, and abnormal results in many routine laboratory tests; however, obesity, Black race, Hispanic ethnicity, chronic obstructive pulmonary disease, hypertension, and smoking were not associated with mortality.In this study, most deaths from SARS-CoV-2 occurred in patients with age of 50 years or older, male sex, and greater comorbidity burden. This cohort study identifies excess risk and risk factors associated with hospitalization, mechanical ventilation, and mortality in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Identifying independent risk factors for adverse outcomes in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can support prognostication, resource utilization, and treatment.To identify excess risk and risk factors associated with hospitalization, mechanical ventilation, and mortality in patients with SARS-CoV-2 infection.This longitudinal cohort study included 88 747 patients tested for SARS-CoV-2 nucleic acid by polymerase chain reaction between Feburary 28 and May 14, 2020, and followed up through June 22, 2020, in the Department of Veterans Affairs (VA) national health care system, including 10 131 patients (11.4%) who tested positive.Sociodemographic characteristics, comorbid conditions, symptoms, and laboratory test results.Risk of hospitalization, mechanical ventilation, and death were estimated in time-to-event analyses using Cox proportional hazards models.The 10 131 veterans with SARS-CoV-2 were predominantly male (9221 [91.0%]), with diverse race/ethnicity  and a mean (SD) age of 63.6 (16.2) years. Compared with patients who tested negative for SARS-CoV-2, those who tested positive had higher rates of 30-day hospitalization , mechanical ventilation , and death . Among patients who tested positive for SARS-CoV-2, characteristics significantly associated with mortality included older age , high regional COVID-19 disease burden , higher Charlson comorbidity index score , fever , dyspnea , and abnormalities in the certain blood tests, which exhibited dose-response associations with mortality, including aspartate aminotransferase , creatinine , and neutrophil to lymphocyte ratio . With the exception of geographic region, the same covariates were independently associated with mechanical ventilation along with Black race , male sex , diabetes , and hypertension . Notable characteristics that were not significantly associated with mortality in adjusted analyses included obesity , Black race , Hispanic ethnicity , chronic obstructive pulmonary disease , hypertension , and smoking . Most deaths in this cohort occurred in patients with age of 50 years or older (63.4%), male sex (12.3%), and Charlson Comorbidity Index score of at least 1 (11.1%).In this national cohort of VA patients, most SARS-CoV-2 deaths were associated with older age, male sex, and comorbidity burden. Many factors previously reported to be associated with mortality in smaller studies were not confirmed, such as obesity, Black race, Hispanic ethnicity, chronic obstructive pulmonary disease, hypertension, and smoking. Most prior studies have been local or regional, rather than national, in scope. Finally, most studies have not compared patients who tested positive for SARS-CoV-2 with those who tested negative to determine the excess risk associated with SARS-CoV-2 infection itself as opposed to other underlying comorbid conditions in patients who happen to have SARS-CoV-2 infection. To address this knowledge gap, we used national data from the Department of Veterans Affairs (VA) health care system to determine the risk of hospitalization, mechanical ventilation, and death associated with infection and to identify characteristics independently associated with these outcomes in patients who tested positive for SARS-CoV-2.Multiple risk factors for developing severe COVID-19 disease have been reported, including sociodemographic factors and comorbid conditions.25 which includes analytic variables extracted from the Corporate Data Warehouse for all VA enrollees tested for SARS-CoV-2. Using this resource, we identified all VA enrollees (N = 88 747) who had nasopharyngeal swabs tested for SARS-CoV-2 nucleic acid by polymerase chain reaction in inpatient or outpatient VA facilities (including VA nursing homes) between February 28 and May 14, 2020, for any indication, excluding those who were VA employees. Most tests were performed in VA laboratories using the US Food and Drug Administration–approved RealTime (Abbott Laboratories) or Xpert-Xpress (Cepheid) SARS-CoV-2 assays; some were sent to commercial or state public health laboratories, especially during the early days of the pandemic. Cohort members were followed up through June 22, 2020, for study outcomes allowing for a minimum follow-up of 39 days. This study was approved by the institutional review board of the Veterans Affairs Puget Sound Healthcare System, which granted a waiver of informed consent because this was a retrospective cohort study based on an existing database. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.The VA supports the largest integrated national health care system in the United States and provides care for more than 6 million veterans annually. The VA uses a single, national comprehensive electronic health care information network. We derived data from the VA’s Corporate Data Warehouse, a data repository of electronic medical records, developed by the VA Informatics and Computing Infrastructure (VINCI) to facilitate research. To support COVID-19 research, VINCI analysts created and are continually updating the COVID-19 Shared Data Resource,index date for all analyses was defined as the date of the earliest positive test (for patients with SARS-CoV-2) or the date of the earliest negative test (for patients with no SARS-CoV-2), unless the patient had been admitted to a VA hospital during the preceding 15 days, in which case the date of admission served as the index date.Patients were defined as positive for SARS-CoV-2 if they had at least 1 positive polymerase chain reaction test (n = 10 131 [11.4%]) during the ascertainment period. Patients were defined as negative for SARS-CoV-2 if all polymerase chain reaction tests were negative (n = 78 616 [88.6%]). Final adjudication of SARS-CoV-2 status was performed by the VA National Surveillance Tool, which is intended to be the single, authoritative data source for the determination of positive and negative cases within the Veterans Health Administration. The 26 Episodes of mechanical ventilation and hospitalization that occurred outside the VA were captured only if the VA paid for these hospitalizations at non-VA facilities under the VA Community Care program.We determined the following 3 outcomes: (1) hospitalization at the index date or within 15 days of the index date, (2) mechanical ventilation at the index date or within 60 days, and (3) all-cause mortality at any time after the index date. Deaths that occurred both in and out of the VA are comprehensively captured in the Corporate Data Warehouse through a variety of sources, including VA inpatient files, VA Beneficiary Identification and Records Locator System, Social Security Administration death files, and the Department of Defense.27We included characteristics that were evaluated in prior studies or that we hypothesized might be associated with adverse outcomes. Baseline sociodemographic characteristics included age, sex, race, ethnicity, body mass index , urban vs rural residence (based on zip codes), and regional per capita COVID-19–related mortality, operationalized as the number of COVID-19–related deaths per million residents in each participant’s state of residence as of June 11, 2020 .27International Classification of Diseases and Related Health Problems, Tenth Revision (ICD-10) recorded in VA electronic health records during the 2-year period on or before the index date.25 We used the Charlson Comorbidity Index (CCI) to estimate the overall burden of comorbidity  for each major risk factor were estimated by finding the mean over randomly selected permutations of the PAF when other risk factors were sequentially removed from the model. The number of permutations was sufficient to approximate the true mean to within 0.1%. Confidence intervals were calculated using Monte Carlo simulation (500 iterations over 5000 samples).P < .05, and all tests were 2-tailed.Analysis was conducted in Stata MP version 15 (StataCorp), R 64-bit version 3.6.1 , with the averisk package version 1.0.3. Statistical significance was set at Of 88 747 VA enrollees tested for SARS-CoV-2, 10 131 (11.4%) tested positive , A. CompCompared with individuals who tested negative for SARS-CoV-2, those who tested positive had higher 30-day rates of hospitalization , mechanical ventilation , and mortality  after adjusting for sociodemographic characteristics and comorbid conditions . CompetiVeterans who tested positive for SARS-CoV-2 had a mean (SD) age of 63.6 (16.2) years; 9221 (91.0%) were men, 944 (9.3%) were Hispanic individuals, 5022 (49.6%) were White individuals, and 4215 (41.6%) were Black individuals . They weIncreasing age was the characteristic most strongly associated with risk of hospitalization, mechanical ventilation, and death. Compared with patients younger than 50 years of age , those aged 50 to 64 years , 65 to 79 years , and 80 years and older  had progressively higher mortality , C. CompVeterans who tested positive for SARS-CoV-2 had a high overall burden of comorbidity , with leThe most common documented symptoms included fever (4187 [41.3%]), cough (2620 [25.9%]), and dyspnea (1907 [18.8%]) . Symptom9, multiply by 0.001], mechanical ventilation: aHR, 2.34; 95% CI, 1.74-3.14; mortality: aHR, 2.16; 95% CI, 1.62-2.87), elevated neutrophil count , reduced lymphocyte count , reduced albumin , and elevated alanine aminotransferase  , elevated serum aspartate aminotransferase , elevated neutrophil to lymphocyte ratio , elevated total white blood cell count (>11 200/μL vs ≤4770/μL [to convert to ×1035-2.57) , but not35-2.57) .Most deaths (63.4%) were associated with older age groups relative to the reference group : 6.2%  were associated with age 50 to 64 years, 28.9%  with age 65 to 79 years, and 28.3%  with age of 80 years or older , D. MaleIn a national study of 88 747 US veterans tested for SARS-CoV-2 infection between February 28 and May 14, 2020, those testing positive had a 4.2-fold risk of mechanical ventilation and a 4.4-fold risk of death compared with those testing negative. Among those who tested positive for SARS-CoV-2, older age was the strongest risk factor associated with hospitalization, mechanical ventilation, and mortality. Most deaths in this cohort were attributed to age of 50 years or older (63.4%), male sex (12.3%), and comorbidity burden, with CCI score of at least 1 (11.1%). Other risk factors for mortality included select preexisting comorbid conditions  and presenting symptoms . Abnormal results in a range of routine laboratory tests were associated with mechanical ventilation or mortality in a dose-response manner.2 Within health systems, the percentage of patients who have been hospitalized ranged from 8% to 80.7%, depending on the clinical context of testing.32 The percentages of patients who require mechanical ventilation has ranged from 2.3% of the Chinese population to 93.2% of critically ill patients infected with SARS-CoV-2 admitted to New York area hospitals.46 Short-term mortality rates in the US population are estimated to be between 1.8% and 3.4%,2 which is higher than the 1.4% estimate from China earlier in the pandemic.33 However, short-term mortality rates in case series of hospitalized patients and high-risk populations have been much higher, ranging from 10.2% to 67%.51 Our findings demonstrating 30-day rates of hospitalization, mechanical ventilation, and death of 30.4%, 6.7%, and 10.8%, respectively, spotlight the substantial consequences of SARS-CoV-2 on the Veteran population, associated with the high prevalence of advanced age, male sex, and comorbid conditions.Early estimates from the US Centers for Disease Control and Prevention suggested that 20.7% to 31.4% of US adults infected with SARS-CoV-2 were hospitalized.24Recognizing risk factors for adverse outcomes is a preliminary step toward developing prognostic models that will allow for real-time identification of patients most and least likely to benefit from available interventions . Some risk factors may be reversible or modifiable, such that eliminating them might be a strategy for reducing the mortality rate of SARS-CoV-2 or may provide clues as to the pathogenesis of severe, life-threatening SARS-CoV-2. Risk factors that have been identified in prior studies include older age, male sex, hypertension, diabetes, chronic obstructive pulmonary disease, cardiac disease, liver disease, chronic kidney disease, neurologic disorders, cancer, obesity, higher overall burden of comorbidity, and smoking.In our cohort, older age was by far the strongest risk factor associated with ventilation and death, even after adjusting for comorbid conditions; 63.5% of deaths were attributed to being aged 50 years or older based in PAF calculations. While we observed linear associations between age and mortality, the association of age with mechanical ventilation appeared to be nonlinear, with the highest risk noted for those aged 65 to 79 years, perhaps reflecting treatment preferences and/or clinical practice. We observed strong linear associations with CCI score and all measured outcomes, suggesting that a measure of overall disease burden may be more helpful than the presence of individual comorbid conditions. PAF calculations suggested that 11.1% of deaths were attributed to having a CCI score of at least 1. Although male sex was not statistically significantly associated with mortality, 12.3% of deaths were attributed to male sex based on PAF calculations, which were statistically significant. Among hospitalized patients, abnormalities in a range of routine laboratory tests were strongly and linearly associated with ventilation and death, suggesting that these could be useful in risk stratification at the time of hospitalization.52 Additionally, we investigated risk factors for adverse outcomes among patients who tested positive for SARS-CoV-2 rather than in the general population53 because the latter approach provides a composite of the risk of infection and subsequent risk of death. For example, Black and Hispanic patients may be much more likely to acquire SARS-CoV-2 but not more likely to die if infected. The finding that Black patients did not have higher mortality rates but had higher mechanical ventilation rates may be related to lower rates of advance directives in Black patients.54Some risk factors for mortality reported by earlier studies did not reach statistical significance in our analyses including Black race, Hispanic ethnicity, body mass index, underlying lung disease, smoking, diabetes, and hypertension. This may reflect differences in the study population , differences in the confounders that were adjusted for, or attenuation of racial/ethnic disparities in access to care in the VA system relative to the private sector.Surprisingly, neither chronic obstructive pulmonary disease nor smoking—which are prevalent in the veteran population—were associated with adverse outcomes. Geographic burden of SARS-CoV-2 was not as strongly associated with mortality as we had anticipated. VA patients in the most highly affected states  only had a 1.21-fold higher mortality rate than patients from the least affected states.ICD-10 codes for the determination of comorbid conditions. However, most ICD-10–based definitions have been widely used and validated in VA studies. Novel natural language processing plus ICD-10 codes were used for the definition of SARS-CoV-2 symptoms, although the performance characteristics of these definitions are not yet known. We captured deaths that occurred both within and outside the VA; however, hospitalizations or mechanical ventilations that occurred outside the VA and were not paid for by the VA were not captured. Our results are limited to those patients who were tested within the VA system. Therefore, our results likely reflect institutional policies and practices related to testing. Strengths of our study include its national scope, large number of patients, relatively long follow-up for a range of outcomes, and analysis of many potential risk factors.This study has limitations. Our results in the predominantly male veteran population may not be generalizable to other populations and groups, especially women. We used In this study, we found high rates of mechanical ventilation and death among 10 131 VA patients with SARS-CoV-2 infection. Most deaths were associated with older age, male sex, and a high overall burden of comorbidity.


Mucopolysaccharidoses (MPSs) are inherited disorders of the glycosaminoglycan (GAG) metabolism. The defective digestion of GAGs within the intralysosomal compartment of affected patients leads to a broad spectrum of clinical manifestations ranging from cardiovascular disease to neurological impairment. The molecular mechanisms underlying the progression of the disease downstream of the genetic mutation of genes encoding for lysosomal enzymes still remain unclear. Here, we applied a targeted metabolomic approach to a mouse model of PS IIIB, using a platform dedicated to the diagnosis of inherited metabolic disorders, in order to identify amino acid and fatty acid metabolic pathway alterations or the manifestations of other metabolic phenotypes. Our analysis highlighted an increase in the levels of branched-chain amino acids , aromatic amino acids (Tyr and Phe), free carnitine, and acylcarnitines in the liver and heart tissues of MPS IIIB mice as compared to the wild type (WT). Moreover, Ala, Met, Glu, Gly, Arg, Orn, and Cit amino acids were also found upregulated in the liver of MPS IIIB mice. These findings show a specific impairment of the BCAA and fatty acid catabolism in the heart of MPS IIIB mice. In the liver of affected mice, the glucose-alanine cycle and urea cycle resulted in being altered alongside a deregulation of the BCAA metabolism. Thus, our data demonstrate that an accumulation of BCAAs occurs secondary to lysosomal GAG storage, in both the liver and the heart of MPS IIIB mice. Since BCAAs regulate the biogenesis of lysosomes and autophagy mechanisms through mTOR signaling, impacting on lipid metabolism, this condition might contribute to the progression of the MPS IIIB disease. Lysosomal storage diseases (LSDs) are a group of hereditary metabolic disorders which are inherited in an autosomal recessive manner, except for some that are X-linked . They arAmong the LSDs, the mucopolysaccharidoses (MPSs) are eleven inherited metabolic diseases caused by a deficiency of the lysosomal enzymes needed to catabolize glycosaminoglycans (GAGs) . A progrn-acetylglucosaminidase  enzyme involved in the degradation of heparan sulfate (HS) is one of the four MPS III (Sanfilippo syndrome) subtypes [NAGLU gene [The autosomal recessive disorder MPS IIIB caused by mutations in the gene encoding for the α-subtypes . Patientsubtypes . A murinGLU gene . These mGLU gene . HoweverGLU gene ,23,24,25−/− mouse model of the MPS IIIB disease. The analysis of the metabolite profile and the identification of the main impaired metabolic processes affected by the absence of NAGLU in the heart and liver tissues of mice might provide further insights into the molecular mechanisms underlying the onset and progression of the disease, thus helping the search for an effective cure of such intractable disease.Metabolomics is an emerging “omics” science that represents an appealing and useful tool to determine changes in the profile of metabolites in living systems resulting from environmental conditions and/or genetic backgrounds ,27,28. T−/− mice compared to age-matched wild-type (WT) mice.A targeted metabolic analysis was performed to evaluate changes in the concentration of Ala, Val, Xle (Leu and/or Ile), Met, Phe, Tyr, Asp, Glu, Gly, Orn, Cit, and Arg amino acids and saturated, unsaturated, hydroxylated and branched acylcarnitines in the heart and liver of NAGLUFirstly, in order to test the power of the comparative studies performed, we carried out Spearman’s rank correlation analysis to evaluate sample reproducibility within the groups based on nine animals for each genotype for each tissue. As expected, the overall analysis displayed a positive intragroup correlation and a negative intergroup correlation in both the analyzed tissue-metabolomes a,b.−/− and WT) in the metabolic profiles characterizing the heart and liver. The PCA score plot revealed a clear separation between the analyzed data groups for each tissue  1 variance 40.8%, PC2 variance 21.9%; liver: PC1 variance 44.0%, PC2 variance 9%). Outliers were not found in the heart and liver metabolomes . The results showed correct sample group clusterization, and the dendrogram structure, using Euclidean distance, resulted in two main clusters, relating to the compared samples (NAGLU−/− and WT) and metabolites, respectively . Thus, the detected metabolic abnormalities are likely to be associated with the MPS IIIB phenotype, allowing the identification of a pathological metabolomic signature of the MPS IIIB disease.With a univariate analysis, we detected significant differences in bot amino acid and acylcarnitine levels in the liver and heart of MPS IIIB mice when compared to WT.−/− mice when compared to WT, although to a lesser extent in the heart  score. From the PLS-DA, the differentiation between NAGLU−/− and WT genotypes occurred mainly along component 1, measured as 34.2% and 44.1% in the heart and liver tissues, respectively. The most important features identified by the PLS-DA and the VIP score plot (threshold > 1.5) included six metabolites in the heart, C5:1, C6:1, C4, C18:1, C16:1, and C10:2, and eight key metabolites in the liver, C4, C12OH, C3, C6DC, C4OH, C8, C6, and C4DC, as potentially closely associated with NAGLU deficiency  was performed , Phe and Tyr amino acids, free carnitine, and acylcarnitines in both liver and heart tissue. The augmented levels of BCAAs concomitant with the accumulation of short-chain acylcarnitines , involved in BCAA metabolism [The present study shows that NAGLUtabolism ,34, suggtabolism . FurtherIn the liver tissue of MPS IIIB mice, as well as the amino acids discussed above, we also found elevated levels of the Ala, Met, Glu, Gly, Arg, Orn, and Cit amino acids. These results suggest a deregulation of the glucose-alanine cycle and the urea cycle in the liver of MPS IIIB mice, as well as the altered BCAA and other amino acid metabolisms. These results are consistent with a previous metabolite analysis of the liver of MPS I mice showing decreased levels of lipids, simple carbohydrates, and nucleotides, whereas most amino acids, amino acid derivatives, dipeptides, and urea resulted in being increased, suggesting an increase in protein catabolism . AlthougThe enhancement of protein catabolism in MPS IIIB mouse tissue at least in part may fulfill the intermediary metabolism in response to carbohydrate and lipid deficiencies. Indeed, inside the cells, BCAAs can be stored in amino acid pools, integrated into proteins, or shuttled to mitochondria for oxidation. They serve as signaling molecules regulating glucose and lipid metabolism as well as protein synthesis through the phosphoinositide 3-kinase/protein kinase B/mammalian target of the rapamycin (PI3K/AKT/mTOR) signal pathway ,39. In pMultiple reports show that an impairment of autophagy by engulfed lysosomes drives the progression of the disease in LSDs ,43,44,45−/− mouse heart correlates well with our previous findings demonstrating an impaired lysosomal autophagic flux associated with cardiac phenotype in MPS IIIB mice [Amino acids promote the localization of mTORC1 at the lysosomes and induce its activation through both Rag-dependent and Rag-independent pathways ,53,54. LIIB mice .Notably, lysosomal mTORC1 and Rag GTPases have been shown to regulate the localization of the transcription factor EB (TFEB) involved in the lysosomal biogenesis and autophagy, by phosphorylating it on several serine and threonine residues . When phAn impairment of autophagy is usually associated with the dysregulation of mitochondrial quality control pathways and the accumulation of damaged mitochondria within the cells. In LSDs, reduced autophagic flux has been shown to lead to the persistence of dysfunctional mitochondria with a consequent increase in reactive oxygen species production, altered calcium homeostasis, and enhanced pro-apoptotic signals ,61,62. MIn conclusion, the metabolic phenotype observed in the liver and heart of MPS IIIB mice supports the view that autophagy and mitophagy deregulation may play a prominent role in the pathogenesis of the MPS IIIB disease. This may be relevant for the development of effective therapies for curing MPS IIIB.−/−) was created by Prof. Elizabeth Neufeld, UCLA, by interrupting exon 6 of the NAGLU gene on the C57/BL6 background [−/− and WT mice were genotyped by PCR [The animal model of MPS IIIB  analysis was performed according to published methods, with little modification ,70. The 15N, 2-13C-Gly, 2H4-Ala, 2H8-Val, 2H3-Leu, 2H3-Met, 13C6-Phe, 13C6-Tyr, 2H3-Asp, 2H3-Glu, 2H2-Orn, 2H2-Cit , and 2H4,5-13C-Arg and 2H9-C0, 2H3-C2, 2H3-C3, 2H3-C4, 2H9-C5, 2H3-C8, 2H9-C14, and 2H3-C16 , respectively. The samples were shaken for 20 min at room temperature on an orbital shaking system and dried under a nitrogen flow at 40 °C. AAs and ACs were derivatized to butyl esters with 80 μL of butanol in 3N HCl  at 65 °C for 25 min. The samples were dried again after derivatization and were, finally, resuspended in 300 μL of acetonitrile/water (70:30) containing 0.1% formic acid. For the MS/MS experiments, 60 μL of the final solution was injected in flow injection analysis mode. Each sample was analyzed in triplicate.A total of 100 µL of the standard mixture containing labeled AAs and ACs were added to 100 µL of the sample. The standard concentrations were in the 500–2500 μmol/L range for AAs and in the 7.6–152 μmol/L range for ACs. The labeled AA and AC mixtures were composed of A group of metabolites constituted of 12 AAs and 35 ACs was measured employing a targeted MS-based metabolomic platform . In detaMS/MS analysis for Ala, Val, Xle, Met, Phe, Tyr, Asp, and Glu was conducted using a neutral loss of 102 Da scan function, according to the following parameters: polarity: positive; m/z range (Da): 130–280; declustering potential (DP) (volts): 45; collision energy (CE) (volts): 25. MS/MS analysis for Gly, Orn, Cyt, and Arg was performed in multiple reaction monitoring (MRM) mode, according to the following parameters: polarity: positive; Q1/Q3 (m/z): 132.1/76.0 (Gly), 189.1/70.0 (Orn), 231.2/70.0 (Arg), 232.2/113.1 (Cit); DP (volts): 43 (Gly), 33 (Orn), 60 (Arg), 50 (Cit); CE (volts): 14 (Gly), 33 (Orn), 45 (Arg), 28 (Cit).Finally, a precursor scan of 85.1 Da was used for the ACs analysis, according to the following parameters: polarity: positive; m/z range (Da): 200–560; DP range (volts): 55–80; CE range (volts): 34–60.www.erndimqa.nl), prepared at low, mid, high, and very high concentrations.Quantitative analysis of the data was performed with ChemoView v1.2 software through the comparison of the analyte and its corresponding internal standard areas. Using stable isotope-labeled internal standards improves correction for the matrix effect. The MS method’s accuracy and precision were estimated by analyzing quality control (QC) samples provided by ERNDIM  was evaluated by parametric (unpaired t-test) or non-parametric (Mann–Whitney test) tests when data failed the Shapiro–Wilk normality test. Two outlier samples were removed from the metabolome analysis.Univariate statistics were calculated with GraphPad Prism 8.0, and the results are presented as the mean ± standard error of the mean (SEM). The statistical significance of the difference in metabolite tissue concentrations between genotype conditions  [http://www.metaboanalyst.ca). The Human Metabolome Database was used to assign the correctly numbered ID (HMDB ID) to each differentially abundant metabolite. Multivariate statistical analysis and metabolite set enrichment analysis were performed using MetaboAnalyst 4.0 (lyst.ca) . The norIn summary, targeted metabolomic profiling demonstrates elevated levels of BCAAs, other amino acids, and acylcarnitines in the liver and heart of the MPS IIIB mouse model. We also identified impaired metabolic pathways such as BCAA catabolism and phenylalanine and tyrosine metabolism in both the liver and heart; branched- and long-chain fatty acid β-oxidation in the heart; and mainly the urea cycle and the glucose-alanine cycle in the liver of MPS IIIB mice. We speculate that the secondary accumulation of BCAAs and other amino acids in the liver and the heart of MPS IIIB mice affects autophagy, which in turn leads to altered cellular metabolism, thus contributing to the progression of the disease.