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Antibody-mediated autoimmune pathologies like membranous nephropathy are difficult to model, particularly in the absence of local target antigen expression in model organisms such as mice and rats; as is the case for phospholipase A2 receptor 1 (PLA2R1), the major autoantigen in membranous nephropathy. Here, we generated a transgenic mouse line expressing the full-length human PLA2R1 in podocytes, which has no kidney impairment after birth. Beginning from the age of three weeks, these mice spontaneously developed anti-human PLA2R1 antibodies, a nephrotic syndrome with progressive albuminuria and hyperlipidemia, and the typical morphological signs of membranous nephropathy with granular glomerular deposition of murine IgG in immunofluorescence and subepithelial electron-dense deposits by electron microscopy. Importantly, human PLA2R1-expressing Rag2

Glomerular disease due to podocyte malfunction is a major factor in the pathogenesis of chronic kidney disease. Identification of podocyte-specific signaling pathways is therefore a prerequisite to characterizing relevant disease pathways and developing novel treatment approaches. Here, we employed loss of function studies for EPB41L5 (Yurt) as a central podocyte gene to generate a cell type-specific disease model. Loss of Yurt in fly nephrocytes caused protein uptake and slit diaphragm defects. Transcriptomic and proteomic analysis of human EPB41L5 knockout podocytes demonstrated impaired mechanotransduction via the YAP/TAZ signaling pathway. Further analysis of specific inhibition of the YAP/TAZ-TEAD transcription factor complex by TEADi led to the identification of ARGHAP29 as an EPB41L5 and YAP/TAZ-dependently expressed podocyte RhoGAP. Knockdown of ARHGAP29 caused increased RhoA activation, defective lamellipodia formation, and increased maturation of integrin adhesion complexes, explaining similar phenotypes caused by loss of EPB41L5 and TEADi expression in podocytes. Detection of increased levels of ARHGAP29 in early disease stages of human glomerular disease implies a novel negative feedback loop for mechanotransductive RhoA—YAP/TAZ signaling in podocyte physiology and disease.

Thrombospondin type-1 domain-containing 7A (THSD7A) is a large extracellular protein that is found in podocyte foot processes of the kidney glomerulus. It has been established as a causative autoantigen in membranous nephropathy. Amongst the predicted 21 thrombospondin repeat domains of its extracellular segment, the highest frequency of autoimmune response has been associated with the two N-terminal domains. Here, we show that antibodies against this THSD7A segment in mice induce typical clinical and morphological signs of membranous nephropathy. The high-resolution structure of these two domains reveals a non-canonical thrombospondin repeat fold that is distinct from the established type 1 thrombospondin repeat. As it shares a conserved disulfide pattern with the canonical fold, we refer to these domains d1 and d2 as type 1A thrombospondin repeats. Both domains comprise a seven layered CC-W-PP-R-W-QQ-CC pattern, which is only partly shared by other THSD7A thrombospondin repeat domains. The two domains form a well-defined V-shaped tandem arrangement. Our findings provide crucial insight into specific structural features of these two domains that are distinct from other regions of THSD7A and hence could cause the high level of antigenicity found for these two domains.

Extracellular vesicles (EVs) are lipid bilayer-enclosed structures that represent newly discovered means for cell-to-cell communication as well as promising disease biomarkers and therapeutic tools. Apart from proteins, lipids, and metabolites, EVs can deliver genetic information such as mRNA, eliciting a response in the recipient cells. In the present study, we have analyzed the mRNA content of brain-derived EVs (BDEVs) isolated 72 h after experimental stroke in mice and compared them to controls (shams) using nCounter® Nanostring panels, with or without prior RNA isolation. We found that both panels show similar results when comparing upregulated mRNAs in stroke. Notably, the highest upregulated mRNAs were related to processes of stress and immune system responses, but also to anatomical structure development, cell differentiation, and extracellular matrix organization, thus indicating that regenerative mechanisms already take place at this time-point. The five top overrepresented mRNAs in stroke mice were confirmed by RT-qPCR and, interestingly, found to be full-length. We could reveal that the majority of the mRNA cargo in BDEVs was of microglial origin and predominantly present in small BDEVs (≤ 200 nm in diameter). However, the EV population with the highest increase in the total BDEVs pool at 72 h after stroke was of oligodendrocytic origin. Our study shows that nCounter® panels are a good tool to study mRNA content in tissue-derived EVs as they can be carried out even without previous mRNA isolation, and that the mRNA cargo of BDEVs indicates a possible participation in inflammatory but also recovery processes after stroke.

Vascular endothelial growth factor A (VEGFA) secretion from podocytes is crucial for maintaining endothelial integrity within the glomerular filtration barrier. However, until now, the molecular mechanisms underlying podocyte secretory function remained unclear. Through podocyte-specific deletion of BECLIN1 (ATG6 or Becn1), a key protein in autophagy initiation, we identified a major role for this molecule in anterograde Golgi trafficking. The Becn1-deficient podocytes displayed aberrant vesicle formation in the trans-Golgi network (TGN), leading to dramatic vesicle accumulation and complex disrupted patterns of intracellular vesicle trafficking and membrane dynamics. Phenotypically, podocyte-specific deletion of Becn1 resulted in early-onset glomerulosclerosis, which rapidly progressed and dramatically reduced mouse life span. Further, in vivo and in vitro studies clearly showed that VEGFA secretion, and thereby endothelial integrity, greatly depended on BECLIN1 availability and function. Being the first to demonstrate the importance of a secretory pathway for podocyte integrity and function, we identified BECLIN1 as a key component in this complex cellular process. Functionally, by promoting VEGFA secretion, a specific secretory pathway emerged as an essential component for the podocyte-endothelial crosstalk that maintains the glomerular filtration barrier.

OBJECTIVE: Parkinson's disease (PD) manifests in motor dysfunction, non-motor symptoms, and eventual dementia (PDD). Neuropathological hallmarks include nigrostriatal neurodegeneration, Lewy body (LB) pathology, and neuroinflammation. Alpha-synuclein (α-syn), a primary component of LBs, is implicated in PD pathogenesis, accumulating and aggregating in both familial and sporadic PD. However, as α-syn pathology is often comorbid with amyloid-beta (Aβ) plaques and phosphorylated tau (pTau) tangles in PDD, it is still unclear whether α-syn is the primary cause of neurodegeneration in sporadic PDD. We aimed to determine how the absence of α-syn would affect PDD manifestation.METHODS: IFN-β knockout (Ifnb-/- ) mice spontaneously develop progressive behavior abnormalities and neuropathology resembling PDD, notably with α-syn+ LBs. We generated Ifnb/Snca double knockout (DKO) mice and evaluated their behavior and neuropathology compared with wild-type (Wt), Ifnb-/- , and Snca-/- mice using immunohistochemistry, electron microscopy, immunoblots, qPCR, and modification of neuronal signaling.RESULTS: Ifnb/Snca DKO mice developed all clinical PDD-like behavioral manifestations induced by IFN-β loss. Independently of α-syn expression, lack of IFN-β alone induced Aβ plaques, pTau tangles, and LB-like Aβ+ /pTau+ inclusion bodies and neuroinflammation. IFN-β loss caused significant elevated glial and neuronal TNF-α and neuronal TNFR1, associated with neurodegeneration. Restoring neuronal IFN-β signaling or blocking TNFR1 rescued caspase 3/t-BID-mediated neuronal-death through upregulation of c-FLIPS and lowered intraneuronal Aβ and pTau accumulation.INTERPRETATION: These findings increase our understanding of PD pathology and suggest that targeting α-syn alone is not sufficient to mitigate disease. Targeting specific aspects of neuroinflammation, such as aberrant neuronal TNF-α/TNFR1 or IFN-β/IFNAR signaling, may attenuate disease. This article is protected by copyright. All rights reserved.

cited By 1 Skeletal muscle wasting is commonly associated with chronic kidney disease (CKD), resulting in increased morbidity and mortality. However, the link between kidney and muscle function remains poorly understood. Here, we took a complementary interorgan approach to investigate skeletal muscle wasting in CKD. We identified increased production and elevated blood levels of soluble pro-cachectic factors, including activin A, directly linking experimental and human CKD to skeletal muscle wasting programs. Single-cell sequencing data identified the expression of activin A in specific kidney cell populations of fibroblasts and cells of the juxtaglomerular apparatus. We propose that persistent and increased kidney production of procachectic factors, combined with a lack of kidney clearance, facilitates a vicious kidney/muscle signaling cycle, leading to exacerbated blood accumulation and, thereby, skeletal muscle wasting. Systemic pharmacological blockade of activin A using soluble activin receptor type IIB ligand trap as well as muscle-specific adeno-associated virus-mediated downregulation of its receptor ACVR2A/B prevented muscle wasting in different mouse models of experimental CKD, suggesting that activin A is a key factor in CKD-induced cachexia. In summary, we uncovered a crosstalk between kidney and muscle and propose modulation of activin signaling as a potential therapeutic strategy for skeletal muscle wasting in CKD.

A growing body of evidence suggests that low nephron numbers at birth can increase the risk of chronic kidney disease or hypertension later in life. Environmental stressors, such as maternal malnutrition, medication and smoking, can influence renal size at birth. Using metanephric organ cultures to model single-variable environmental conditions, models of maternal disease were evaluated for patterns of developmental impairment. While hyperthermia had limited effects on renal development, fetal iron deficiency was associated with severe impairment of renal growth and nephrogenesis with an all-proximal phenotype. Culturing kidney explants under high glucose conditions led to cellular and transcriptomic changes resembling human diabetic nephropathy. Short-term high glucose culture conditions were sufficient for long-term alterations in DNA methylation-associated epigenetic memory. Finally, the role of epigenetic modifiers in renal development was tested using a small compound library. Among the selected epigenetic inhibitors, various compounds elicited an effect on renal growth, such as HDAC (entinostat, TH39), histone demethylase (deferasirox, deferoxamine) and histone methyltransferase (cyproheptadine) inhibitors. Thus, metanephric organ cultures provide a valuable system for studying metabolic conditions and a tool for screening for epigenetic modifiers in renal development.

Background Podocytes embrace the glomerular capillaries with foot processes, which are interconnected by a specialized adherens junction to ultimately form the filtration barrier. Altered adhesion and loss are common features of podocyte injury, which could be mediated by shedding of cell-adhesion molecules through the regulated activity of cell surface-expressed proteases. A Disintegrin and Metalloproteinase 10 (ADAM10) is such a protease known to mediate ectodomain shedding of adhesion molecules, among others. Here we evaluate the involvement of ADAM10 in the process of antibody-induced podocyte injury. Methods Membrane proteomics, immunoblotting, high-resolution microscopy, and immunogold electron microscopy were used to analyze human and murine podocyte ADAM10 expression in health and kidney injury. The functionality of ADAM10 ectodomain shedding for podocyte development and injury was analyzed, in vitro and in vivo, in the anti-podocyte nephritis (APN) model in podocyte-specific, ADAM10-deficient mice. Results ADAM10 is selectively localized at foot processes of murine podocytes and its expression is dispensable for podocyte development. Podocyte ADAM10 expression is induced in the setting of antibody-mediated injury in humans and mice. Podocyte ADAM10 deficiency attenuates the clinical course of APN and preserves the morphologic integrity of podocytes, despite subepithelial immune-deposit formation. Functionally, ADAM10-related ectodomain shedding results in cleavage of the cell-adhesion proteins N- and P-cadherin, thus decreasing their injury-related surface levels. This favors podocyte loss and the activation of downstream signaling events through the Wnt signaling pathway in an ADAM10-dependent manner. Conclusions ADAM10-mediated ectodomain shedding of injury-related cadherins drives podocyte injury.

BACKGROUND: Previous research demonstrated that small Rho GTPases, modulators of the actin cytoskeleton, are drivers of podocyte foot-process effacement in glomerular diseases, such as FSGS. However, a comprehensive understanding of the regulatory networks of small Rho GTPases in podocytes is lacking. METHODS: We conducted an analysis of podocyte transcriptome and proteome datasets for Rho GTPases; mapped in vivo, podocyte-specific Rho GTPase affinity networks; and examined conditional knockout mice and murine disease models targeting Srgap1. To evaluate podocyte foot-process morphology, we used super-resolution microscopy and electron microscopy; in situ proximity ligation assays were used to determine the subcellular localization of the small GTPase-activating protein SRGAP1. We performed functional analysis of CRISPR/Cas9-generated SRGAP1 knockout podocytes in two-dimensional and three-dimensional cultures and quantitative interaction proteomics. RESULTS: We demonstrated SRGAP1 localization to podocyte foot processes in vivo and to cellular protrusions in vitro. Srgap1(fl/fl)*Six2Cre but not Srgap1(fl/fl)*hNPHS2Cre knockout mice developed an FSGS-like phenotype at adulthood. Podocyte-specific deletion of Srgap1 by hNPHS2Cre resulted in increased susceptibility to doxorubicin-induced nephropathy. Detailed analysis demonstrated significant effacement of podocyte foot processes. Furthermore, SRGAP1-knockout podocytes showed excessive protrusion formation and disinhibition of the small Rho GTPase machinery in vitro. Evaluation of a SRGAP1-dependent interactome revealed the involvement of SRGAP1 with protrusive and contractile actin networks. Analysis of glomerular biopsy specimens translated these findings toward human disease by displaying a pronounced redistribution of SRGAP1 in FSGS. CONCLUSIONS: SRGAP1, a podocyte-specific RhoGAP, controls podocyte foot-process architecture by limiting the activity of protrusive, branched actin networks. Therefore, elucidating the complex regulatory small Rho GTPase affinity network points to novel targets for potentially precise intervention in glomerular diseases.

Kidney transplantation is the preferred renal replacement therapy available. Yet, long‐term transplant survival is unsatisfactory, partially due to insufficient possibilities of longitudinal monitoring and understanding of the biological processes after transplantation. Small urinary extracellular vesicles (suEVs) – as a non‐invasive source of information – were collected from 22 living donors and recipients. Unbiased proteomic analysis revealed temporal patterns of suEV protein signature and cellular processes involved in both early response and longer‐term graft adaptation. Complement activation was among the most dynamically regulated components. This unique atlas of the suEV proteome is provided through an online repository allowing dynamic interrogation by the user. Additionally, a correlative analysis identified putative prognostic markers of future allograft function. One of these markers – phosphoenol pyruvate carboxykinase (PCK2) – could be confirmed using targeted MS in an independent validation cohort of 22 additional patients. This study sheds light on the impact of kidney transplantation on urinary extracellular vesicle content and allows the first deduction of early molecular processes in transplant biology. Beyond that our data highlight the potential of suEVs as a source of biomarkers in this setting.

Mutations in chromatin-modifying complexes and metabolic enzymes commonly underlie complex human developmental syn-dromes affecting multiple organs. A major challenge is to determine how disease-causing genetic lesions cause deregulation of homeostasis in unique cell types. Here we show that neural-specific depletion of three members of the non-specific lethal (NSL) chromatin complex—Mof, Kansl2 or Kansl3—unexpectedly leads to severe vascular defects and brain haemorrhaging. Deregulation of the epigenetic landscape induced by the loss of the NSL complex in neural cells causes widespread metabolic defects, including an accumulation of free long-chain fatty acids (LCFAs). Free LCFAs induce a Toll-like receptor 4 (TLR4)–NFκB-dependent pro-inflammatory signalling cascade in neighbouring vascular pericytes that is rescued by TLR4 inhibition. Pericytes display functional changes in response to LCFA-induced activation that result in vascular breakdown. Our work estab-lishes that neurovascular function is determined by the neural metabolic environment.

Apoptotic cell death can be an efficient defense reaction of mammalian cells infected with obligate intracellular pathogens; the host cell dies and the pathogen cannot replicate. While this is well established for viruses, there is little experimental support for such a concept in bacterial infections. All Chlamydiales are obligate intracellular bacteria, and different species infect vastly different hosts. Chlamydia trachomatis infects human epithelial cells; Parachlamydia acanthamoebae replicates in amoebae. We here report that apoptosis impedes growth of P. acanthamoebae in mammalian cells. In HeLa human epithelial cells, P. acanthamoebae infection induced apoptosis, which was inhibited when mitochondrial apoptosis was blocked by codeletion of the mediators of mitochondrial apoptosis, Bax and Bak, by overexpression of Bcl-X(L) or by deletion of the apoptosis initiator Noxa. Deletion of Bax and Bak in mouse macrophages also inhibited apoptosis. Blocking apoptosis permitted growth of P. acanthamoebae in HeLa cells, as measured by fluorescence in situ hybridization, assessment of genome replication and protein synthesis, and the generation of infectious progeny. Coinfection with C. trachomatis inhibited P. acanthamoebae-induced apoptosis, suggesting that the known antiapoptotic activity of C. trachomatis can also block P. acanthamoebae-induced apoptosis. C. trachomatis coinfection could not rescue P. acanthamoebae growth in HeLa; in coinfected cells, C. trachomatis even suppressed the growth of P. acanthamoebae independently of apoptosis, while P. acanthamoebae surprisingly enhanced the growth of C. trachomatis. Our results show that apoptosis can be used in the defense of mammalian cells against obligate intracellular bacteria and suggest that the known antiapoptotic activity of human pathogenic chlamydiae is indeed required to permit their growth in human cells.

International audience; Numerous approaches are being developed to promote post-natal muscle growth based on attenuating Myostatin/Activin signalling for clinical uses such as the treatment neuromuscular diseases, cancer cachexia and sarcopenia. However there have been concerns about the effects of inhibiting Activin on tissues other than skeletal muscle. We intraperitoneally injected mice with the Activin ligand trap, sActRIIB, in young, adult and a progeric mouse model. Treatment at any stage in the life of the mouse rapidly increased muscle mass. However at all stages of life the treatment decreased the weights of the testis. Not only were the testis smaller, but they contained fewer sperm compared to untreated mice. We found that the hypertrophic muscle phenotype was lost after the cessation of sActRIIB treatment but abnormal testis phenotype persisted. In summary, attenuation of Myostatin/Activin signalling inhibited testis development. Future use of molecules based on a similar mode of action to promote muscle growth should be carefully profiled for adverse side-effects on the testis. However the effectiveness of sActRIIB as a modulator of Activin function provides a possible therapeutic strategy to alleviate testicular seminoma development.

AIM: Sodium retention is the hallmark of nephrotic syndrome (NS) and mediated by the proteolytic activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases. Plasmin is highly abundant in nephrotic urine and has been proposed to be the principal serine protease responsible for ENaC activation in NS. However, a proof of the essential role of plasmin in experimental NS is lacking. METHODS: We used a genetic mouse model of NS based on an inducible podocin knockout (Bl6-Nphs2(tm3.1Antc)*Tg(Nphs1-rtTA*3G)(8Jhm)*Tg(tetO-cre)(1Jaw) or nphs2(Δipod)). These mice were crossed with plasminogen deficient mice (Bl6-Plg(tm1Jld) or plg(−/−)) to generate double knockout mice (nphs2(Δipod)*plg(−/−)). NS was induced after oral doxycycline treatment for 14 days and mice were followed for subsequent 14 days. RESULTS: Uninduced nphs2(Δipod)*plg(−/−) mice had normal kidney function and sodium handling. After induction, proteinuria increased similarly in both nphs2(Δipod)*plg(+/+) and nphs2(Δipod)*plg(−/−) mice. Western blot revealed urinary excretion of plasminogen and plasmin in nphs2(Δipod)*plg(+/+) mice which was absent in nphs2(Δipod)*plg(−/−) mice. After onset of proteinuria, amiloride-sensitive natriuresis was increased compared to the uninduced state in both genotypes. Subsequently, urinary sodium excretion dropped in both genotypes leading to an increase in body weight and development of ascites. Treatment with the serine protease inhibitor aprotinin prevented sodium retention in both genotypes. CONCLUSIONS: This study shows that mice lacking urinary plasminogen are not protected from ENaC-mediated sodium retention in experimental NS. This points to an essential role of other urinary serine proteases in the absence of plasminogen.

Summary The mitochondrial proteome is built mainly by import of nuclear-encoded precursors, which are targeted mostly by cleavable presequences. Presequence processing upon import is essential for proteostasis and survival, but the consequences of dysfunctional protein maturation are unknown. We find that impaired presequence processing causes accumulation of precursors inside mitochondria that form aggregates, which escape degradation and unexpectedly do not cause cell death. Instead, cells survive via activation of a mitochondrial unfolded protein response (mtUPR)-like pathway that is triggered very early after precursor accumulation. In contrast to classical stress pathways, this immediate response maintains mitochondrial protein import, membrane potential, and translation through translocation of the nuclear HMG-box transcription factor Rox1 to mitochondria. Rox1 binds mtDNA and performs a TFAM-like function pivotal for transcription and translation. Induction of early mtUPR provides a reversible stress model to mechanistically dissect the initial steps in mtUPR pathways with the stressTFAM Rox1 as the first line of defense., Graphical Abstract, Highlights • Impaired presequence processing leads to precursor aggregation inside mitochondria • Intramitochondrial precursor aggregates trigger early transcriptional stress response • Relocalization of nuclear transcription factor Rox1 to mitochondria ensures survival • Mitochondrial Rox1 maintains mitochondrial genome expression upon early mtUPR, N-terminal presequences direct cytosolic precursor proteins to mitochondria. Poveda-Huertes et al. show that impaired presequence cleavage leads to proteotoxic aggregates inside mitochondria that trigger an early mtUPR-like stress response. Relocalization of the nuclear transcription factor Rox1 to mitochondria allows maintenance of mtDNA expression ensuring proteostasis and survival upon early mtUPR.

Urinary extracellular vesicles (uEVs) have become a promising source for biomarkers accurately reflecting biochemical changes in kidney and urogenital diseases. Characteristically, uEVs are rich in membrane proteins associated with several cellular functions like adhesion, transport, and signaling. Hence, membrane proteins of uEVs should represent an exciting protein class with unique biological properties. In this study, we utilized uEVs to optimize the Triton X-114 detergent partitioning protocol targeted for membrane proteins and proceeded to their subsequent characterization while eliminating effects of Tamm-Horsfall protein, the most abundant interfering protein in urine. This is the first report aiming to enrich and characterize the integral transmembrane proteins present in human urinary vesicles. First, uEVs were enriched using a "hydrostatic filtration dialysis'' appliance, and then the enriched uEVs and lysates were verified by transmission electron microscopy. After using Triton X-114 phase partitioning, we generated an insoluble pellet fraction and aqueous phase (AP) and detergent phase (DP) fractions and analyzed them with LC-MS/MS. Both in- and off-gel protein digestion methods were used to reveal an increased number of membrane proteins of uEVs. After comparing with the identified proteins without phase separation as in our earlier publication, 199 different proteins were detected in DP. Prediction of transmembrane domains (TMDs) from these protein fractions showed that DP had more TMDs than other groups. The analyses of hydrophobicity revealed that the GRAVY score of DP was much higher than those of the other fractions. Furthermore, the analysis of proteins with lipid anchor revealed that DP proteins had more lipid anchors than other fractions. Additionally, KEGG pathway analysis showed that the DP proteins detected participate in endocytosis and signaling, which is consistent with the expected biological functions of membrane proteins. Finally, results of Western blotting confirmed that the membrane protein bands are found in the DP fraction instead of AP. In conclusion, our study validates the use of Triton X-114 phase partitioning protocol on uEVs for a targeted isolation of membrane proteins and to reduce sample complexity. This method successfully facilitates detection of potential biomarkers and druggable targets in uEVs.

The formation of amyloid-like protein structures has recently emerged as a feature in signal transduction, particularly in innate immunity. These structures appear to depend on defined domains for their formation but likely also require dedicated ways to terminate signalling. We, here, define the innate immunity protein/Toll-like receptor adaptor TIR-domain-containing adapter-inducing interferon-β (TRIF) as a novel platform of fibril formation and probe signal initiation through TRIF as well as its termination in Toll-like receptor 3 (TLR3)-stimulated melanoma cells. A main signalling pathway triggered by TLR3 caused apoptosis, which was controlled by inhibitor of apoptosis proteins and was dependent on RIPK1 and independent of TNF. Using correlative electron/fluorescence microscopy, we visualised fibrillar structures formed through both Toll/interleukin-1 receptor and RIP homotypic interacting motif regions of TRIF. We provide evidence that these fibrillary structures are active signalling platforms whose activity is terminated by autophagy. TRIF-signalling enhanced autophagy, and fibrillary structures were partly contained within autophagosomes. Inhibition of autophagy increased levels of pro-apoptotic TRIF complexes, leading to the accumulation of active caspase-8 and enhanced apoptosis while stimulation of autophagy reduced TRIF-dependent death. We conclude that pro-death signals through TRIF are regulated by autophagy and propose that pro-apoptotic signalling through TRIF/RIPK1/caspase-8 occurs in fibrillary platforms.

International audience; Mammalian target of rapamycin (mTOR) signaling is involved in a variety of kidney diseases. Clinical trials administering mTOR inhibitors to patients with FSGS, a prototypic podocyte disease, led to conflicting results, ranging from remission to deterioration of kidney function. Here, we combined complex genetic titration of mTOR complex 1 (mTORC1) levels in murine glomerular disease models, pharmacologic studies, and human studies to precisely delineate the role of mTOR in FSGS. mTORC1 target genes were significantly induced in microdissected glomeruli from both patients with FSGS and a murine FSGS model. Furthermore, a mouse model with constitutive mTORC1 activation closely recapitulated human FSGS. Notably, the complete knockout of mTORC1 by induced deletion of both Raptor alleles accelerated the progression of murine FSGS models. However, lowering mTORC1 signaling by deleting just one Raptor allele ameliorated the progression of glomerulosclerosis. Similarly, low-dose treatment with the mTORC1 inhibitor rapamycin efficiently diminished disease progression. Mechanistically, complete pharmacologic inhibition of mTOR in immortalized podocytes shifted the cellular energy metabolism toward reduced rates of oxidative phosphorylation and anaerobic glycolysis, which correlated with increased production of reactive oxygen species. Together, these data suggest that podocyte injury and loss is commonly followed by adaptive mTOR activation. Prolonged mTOR activation, however, results in a metabolic podocyte reprogramming leading to increased cellular stress and dedifferentiation, thus offering a treatment rationale for incomplete mTOR inhibition.

Kidney transplantation is the preferred renal replacement therapy available. Yet, the biological processes during and after kidney transplantation and how they translate into the overall functional graft outcome are insufficiently understood. Recent developments in the field of extracellular vesicle research allow the deeper exploitation of this non-invasive source. We separated small urinary extracellular vesicles (suEVs) throughout the course of living donor kidney transplantation. SuEVs were collected longitudinally from both the donor and the recipient in 22 living donor kidney transplantations. Unbiased proteomic analysis revealed specific temporal patterns of suEV proteins indicative of the cellular processes involved in the allograft’s response after transplantation with proteins playing a role in complement activation being among the most dynamically regulated components. Using a leave-one-out cross validation model, we identified potential prognostic markers of kidney function at 1 year after transplantation. One of the proteins identified – phosphoenol pyruvate carboxykinase (PCK2) – could be confirmed in an independent validation cohort of another 22 donor-recipient pairs using targeted mass spectrometry. This study sheds the light on early molecular processes during the course of kidney transplantation and shows the future potential of suEVs as a source of biomarkers in this setting. The data set is provided as a unique resource directly accessible through an online tool that allows dynamic interrogation of this first comprising suEV proteome atlas after kidney transplantation.One Sentence SummaryThis study represents the first atlas of the proteomic changes in small urinary extracellular vesicles throughout living donor kidney transplantation identifying PCK2 abundance as a biomarker for renal function 12 months after transplantation

Background The mechanisms balancing proteostasis in glomerular cells are unknown. Mucolipidosis (ML) II and III are rare lysosomal storage disorders associated with mutations of the Golgi-resident GlcNAc-1-phosphotransferase, which generates mannose 6-phosphate residues on lysosomal enzymes. Without this modification, lysosomal enzymes are missorted to the extracellular space, which results in lysosomal dysfunction of many cell types. Patients with MLII present with severe skeletal abnormalities, multisystemic symptoms, and early death; the clinical course in MLIII is less progressive. Despite dysfunction of a major degradative pathway, renal and glomerular involvement is rarely reported, suggesting organ-specific compensatory mechanisms. Methods MLII mice were generated and compared with an established MLIII model to investigate the balance of protein synthesis and degradation, which reflects glomerular integrity. Proteinuria was assessed in patients. High-resolution confocal microscopy and functional assays identified proteins to deduce compensatory modes of balancing proteostasis. Results Patients with MLII but not MLIII exhibited microalbuminuria. MLII mice showed lysosomal enzyme missorting and several skeletal alterations, indicating that they are a useful model. In glomeruli, both MLII and MLIII mice exhibited reduced levels of lysosomal enzymes and enlarged lysosomes with abnormal storage material. Nevertheless, neither model had detectable morphologic or functional glomerular alterations. The models rebalance proteostasis in two ways: MLII mice downregulate protein translation and increase the integrated stress response, whereas MLIII mice upregulate the proteasome system in their glomeruli. Both MLII and MLIII downregulate the protein complex mTORC1 (mammalian target of rapamycin complex 1) signaling, which decreases protein synthesis. Conclusions Severe lysosomal dysfunction leads to microalbuminuria in some patients with mucolipidosis. Mouse models indicate distinct compensatory pathways that balance proteostasis in MLII and MLIII.

Summary: The cellular responses induced by mitochondrial dysfunction remain elusive. Intrigued by the lack of almost any glomerular phenotype in patients with profound renal ischemia, we comprehensively investigated the primary sources of energy of glomerular podocytes. Combining functional measurements of oxygen consumption rates, glomerular metabolite analysis, and determination of mitochondrial density of podocytes in vivo, we demonstrate that anaerobic glycolysis and fermentation of glucose to lactate represent the key energy source of podocytes. Under physiological conditions, we could detect neither a developmental nor late-onset pathological phenotype in podocytes with impaired mitochondrial biogenesis machinery, defective mitochondrial fusion-fission apparatus, or reduced mtDNA stability and transcription caused by podocyte-specific deletion of Pgc-1α, Drp1, or Tfam, respectively. Anaerobic glycolysis represents the predominant metabolic pathway of podocytes. These findings offer a strategy to therapeutically interfere with the enhanced podocyte metabolism in various progressive kidney diseases, such as diabetic nephropathy or focal segmental glomerulosclerosis (FSGS). : Glomerular podocytes form the third and most outer layer of the kidney filtration barrier responsible for restricting the passage of proteins into the urine. Brinkkoetter et al. show that podocyte metabolism primarily relies on anaerobic glycolysis and the fermentation of glucose to lactate. Keywords: glomerular filtration barrier, podocytes, anaerobic glycolysis, metabolomics

Background The mechanisms underpinning the regenerative capabilities of mesenchymal stem cells (MSC) were originally thought to reside in their ability to recognise damaged tissue and to differentiate into specific cell types that would replace defective cells. However, recent work has shown that molecules produced by MSCs (secretome), particularly those packaged in extracellular vesicles (EVs), rather than the cells themselves are responsible for tissue repair. Methods Here we have produced a secretome from adipose-derived mesenchymal stem cells (ADSC) that is free of exogenous molecules by incubation within a saline solution. Various in vitro models were used to evaluate the effects of the secretome on cellular processes that promote tissue regeneration. A cardiotoxin-induced skeletal muscle injury model was used to test the regenerative effects of the whole secretome or isolated extracellular vesicle fraction in vivo. This was followed by bioinformatic analysis of the components of the protein and miRNA content of the secretome and finally compared to a secretome generated from a secondary stem cell source. Results Here we have demonstrated that the secretome from adipose-derived mesenchymal stem cells shows robust effects on cellular processes that promote tissue regeneration. Furthermore, we show that the whole ADSC secretome is capable of enhancing the rate of skeletal muscle regeneration following acute damage. We assessed the efficacy of the total secretome compared with the extracellular vesicle fraction on a number of assays that inform on tissue regeneration and demonstrate that both fractions affect different aspects of the process in vitro and in vivo. Our in vitro, in vivo, and bioinformatic results show that factors that promote regeneration are distributed both within extracellular vesicles and the soluble fraction of the secretome. Conclusions Taken together, our study implies that extracellular vesicles and soluble molecules within ADSC secretome act in a synergistic manner to promote muscle generation. Electronic supplementary material The online version of this article (10.1186/s13287-019-1213-1) contains supplementary material, which is available to authorized users.

Renal proximal tubular cells constantly recycle nutrients to ensure minimal loss of vital substrates into the urine. Although most of the transport mechanisms have been discovered at the molecular level, little is known about the factors regulating these processes. Here, we show that mTORC1 and mTORC2 specifically and synergistically regulate PTC endocytosis and transport processes. Using a conditional mouse genetic approach to disable nonredundant subunits of mTORC1, mTORC2, or both, we showed that mice lacking mTORC1 or mTORC1/mTORC2 but not mTORC2 alone develop a Fanconi-like syndrome of glucosuria, phosphaturia, aminoaciduria, low molecular weight proteinuria, and albuminuria. Interestingly, proteomics and phosphoproteomics of freshly isolated kidney cortex identified either reduced expression or loss of phosphorylation at critical residues of different classes of specific transport proteins. Functionally, this resulted in reduced nutrient transport and a profound perturbation of the endocytic machinery, despite preserved absolute expression of the main scavenger receptors, MEGALIN and CUBILIN. Our findings highlight a novel mTOR–dependent regulatory network for nutrient transport in renal proximal tubular cells.

Activated effector T (TE) cells augment anabolic pathways of metabolism, such as aerobic glycolysis, while memory T (TM) cells engage catabolic pathways, like fatty acid oxidation (FAO). However, signals that drive these differences remain unclear. Mitochondria are metabolic organelles that actively transform their ultrastructure. Therefore, we questioned whether mitochondrial dynamics controls T cell metabolism. We show that TE cells have punctate mitochondria, while TM cells maintain fused networks. The fusion protein Opa1 is required for TM, but not TE cells after infection, and enforcing fusion in TE cells imposes TM cell characteristics and enhances antitumor function. Our data suggest that, by altering cristae morphology, fusion in TM cells configures electron transport chain (ETC) complex associations favoring oxidative phosphorylation (OXPHOS) and FAO, while fission in TE cells leads to cristae expansion, reducing ETC efficiency and promoting aerobic glycolysis. Thus, mitochondrial remodeling is a signaling mechanism that instructs T cell metabolic programming.