Juan Manuel Sacnun

Background: Peritoneal dialysis (PD) effluent of kidney failure patients represents an accessible liquid biopsy of the peritoneal cavity, yet the mechanisms determining its molecular composition remain poorly understood. We applied an integrative multi-omics approach to characterize the composition, transport dynamics, and molecular origin of the PD effluentome. Methods: Cell-free effluent, effluent cells, and plasma were collected from stable PD patients during standardized peritoneal equilibration tests in a randomized clinical trial. Targeted metabolomics, proteomics, and transcriptomic profiling were integrated with a reference human plasma proteome to investigate temporal molecular changes, peritoneal transport characteristics, and protein origin. Results: A total of 207 metabolites and 2970 proteins were identified in PD effluent. Metabolites exhibited distinct class-specific transport kinetics, with rapid equilibration of amino acids and biogenic amines, whereas lipids remained markedly underrepresented despite prolonged dwell times, indicating that transport is governed by physicochemical properties beyond molecular size alone. The effluent proteome underwent concordant alteration, with dwell time-dependent enrichment of pathways related to extracellular matrix organization, angiogenesis, coagulation, and tissue repair. Integrative analysis of the effluent proteome, effluent-cell transcriptome, and human plasma proteome resolved distinct plasma-associated, effluent cell-associated, resident peritoneal tissue-associated, and mixed-origin protein populations. Conclusions: This study establishes the first systems-level approach describing the composition, transport dynamics, and molecular origin of the PD effluentome. By transforming PD effluent into a biologically interpretable molecular readout of peritoneal membrane biology, this work provides a reference for the mechanistic interpretation of effluent-derived biomarkers and supports future therapeutic monitoring and precision medicine in PD.

For patients with kidney failure, peritoneal dialysis (PD) is an alternative kidney replacement therapy to hemodialysis. Long-term PD efficacy is limited by multicellular pathologies induced by the hyperosmotic PD fluids, including vasculopathy and fibroangiogenic remodeling of the peritoneum, which lack targeted pharmacological interventions. We developed a deep secretome profiling workflow in a coculture system using a human peritoneal mesothelial cell line and primary human lymphatic endothelial cells to characterize the influence of PD fluid on communication between these cell types. This approach uncovered extracellular and intracellular signaling patterns absent in standard monocultures and identified 1910 human proteins that revealed extracellular regulatory processes central to PD-associated peritoneal pathology. Integration of the coculture secretome and cellular proteomes with effluent and plasma proteomes derived from patients with PD revealed cell type–specific signaling networks and groups of proteins correlating with clinical parameters such as ultrafiltration capacity, transport kinetics, and inflammation. Functional validation demonstrated that lithium chloride supplementation partially counteracted fibroangiogenic signaling and affected key PD fluid–induced processes, including PDGFC-driven lymphangiogenesis, syndecan-4 ectodomain shedding by endothelial cells, and a mesothelial switch from NOTCH1 to NOTCH3 synthesis. These findings suggest new potential pharmacological strategies to mitigate peritoneal fibrosis and vasculopathy, offering a framework for future therapeutic innovation in PD and beyond.

Background: In systemic sclerosis (SSc), an autoimmune response leads to progressive fibrosis of the skin and internal organs, driven by aberrant activation of fibroblasts. The mechanisms dictating persistent dermal fibroblast (DF) activation and production of extracellular matrix (ECM) remain poorly understood. Nicotinamide N-methyltransferase (NNMT), a SAM-consuming enzyme that modulates cellular methylation potential, has been implicated in fibrotic tissue remodelling in metabolic and malignant diseases. Here, we identify NNMT as a key determinant in DF activation and fibrosis in SSc. Methods: We analyzed bulk, single-cell RNA-Seq and spatial transcriptomics datasets from SSc skin. Functional studies were performed in TGFβ-activated primary human DFs using siRNA-mediated NNMT knockdown (KD) combined with RNA-Seq, metabolite profiling, ELISA, and western blotting. The role of NNMT-regulated transcription factors was assessed by QuantSeq 3′ RNA-Seq following ATF4, SOX9, or SRF KD. Findings: NNMT was markedly upregulated in SSc skin and enriched in disease-expanded SFRP2⁺/COL8A1⁺ myofibroblast states. NNMT KD restored methylation balance by increasing the SAM/SAH ratio and H3K27me3 levels, and abrogated TGFβ-induced profibrotic programs regulating ECM production and collagen synthesis. Mechanistically, NNMT was required for TGFβ-induced upregulation of the transcription factors ATF4, SOX9, and SRF, which together orchestrate ECM gene expression and COL1A1 secretion. Interpretation: These findings define a previously unrecognized TGFβ–NNMT-ATF4/SOX9/SRF axis that coordinates profibrotic transcriptional programs in DFs. Accordingly, NNMT functions as a central effector linking TGFβ signaling to DF activation and ECM remodelling. Targeting NNMT may thus represent a promising therapeutic strategy to attenuate skin fibrosis in SSc.

The peritoneum, pleura, and pericardium are yet understudied multicellular systems where mesothelial (MC) and endothelial cells (EC) are in close proximity. Crosstalk between these cell types likely plays role in molecular transport, immunological reactions and metabolic processes in health, disease, and therapeutic intervention. In this review we discuss recent proteomic efforts to characterize the crosstalk between MC and EC. We describe the proteomic methods necessary for investigation of crosstalk between MC and EC, as well as the in-vitro models that can be employed. Potential experimental approaches range from conditioned medium, via co-culture on semi-permeable membranes to 3D cell culture based organoid models. While the biological and clinical relevance of the models may increase with their ability to mimic close cell communication, the practicality of these complex experiments corresponds vice versa, making standardization more difficult and invoking significant costs. Currently, data and reports on mesothelial-to-endothelial crosstalk are still very scarce. In our opinion, the in-vitro model using semi-permeable cell culture inserts will allow to establish a basic understanding of cellular crosstalk that may occur between those cell types. Later-on more sophisticated 3D cell cultures may be better able to simulate the transport dynamics within the peritoneal membrane.

Used hemodialysis membranes (HD-M) are a valuable reservoir of biological information. Proteins bind to HD-M, but whether this process depends on the type of membrane or patient factors or selectively affects specific protein classes has not been adequately elucidated. State-of-the-art proteomics techniques are capable of identifying and quantifying this therapy-specific subproteome to enable the analysis of disease- or membrane-induced pathophysiologies. We demonstrate the feasibility of the deep proteomic characterization of the extracorporeal proteome adsorbed to HD-M. A shotgun proteomics approach using nano-flow liquid chromatography coupled to mass-spectrometry identified 1648 unique proteins eluted by a chaotropic buffer from the HD-M of eight patients. In total, 995 proteins were present in all eluates; a more stringent approach showed that a core proteome of 310 proteins could be identified independently in all samples. Stability of the dialyzer proteome was demonstrated by a >90% re-identification rate on longitudinal samples of a single patient. The core proteome showed an overrepresentation of pathways of hemostasis and the immune system, and showed differences in membrane materials (polysulfone vs. helixone). This study demonstrates that optimized conditions combined with high-performance proteomics enable the in-depth exploration of the subproteome bound to HD-M, yielding a stable core proteome that can be exploited to study patient-specific factors and improve hemodialysis therapy.

In vitro studies are essential in pre-clinical research. While choice of cell lines is often driven by handling and cost-effectiveness, in-depth knowledge on specific characteristics is scant. Mesothelial cells, which interact with endothelial cells, are widely used in research, including cancer and drug development, but have not been comprehensively profiled. We therefore performed RNA sequencing of polarized, primary peritoneal (HPMC) and immortalized pleural mesothelial cells (MeT-5A), and compared them to endothelial cells from umbilical vein (HUVEC) and cardiac capillaries (HCMEC). Seventy-seven per cent of 12,760 genes were shared between the 4 cell lines, 1003 were mesothelial and 969 were endothelial cell specific. The transcripts reflected major differences between HPMC and MeT-5A in DNA-related processes, extracellular matrix, migration, proliferation, adhesion, transport, growth factor- and immune response, and between HUVEC and HCMEC in DNA replication, extracellular matrix and adhesion organization. Highly variable shared genes were related to six clusters, cell tissue origin and immortalization, but also cell migration capacity, cell adhesion, regulation of angiogenesis and response to hypoxia. Distinct, cell type specific biological processes were further described by cellular component-, molecular function- and Reactome pathway analyses. We provide crucial information on specific features of the most frequently used mesothelial and endothelial cell lines, essential for appropriate use.

To replace kidney function, peritoneal dialysis (PD) utilizes hyperosmotic PD fluids with specific physico-chemical properties. Their composition induces progressive damage of the peritoneum, leading to vasculopathies, decline of membrane function, and PD technique failure. Clinically used PD fluids differ in their composition but still remain bioincompatible. We mapped the molecular pathomechanisms in human endothelial cells induced by the different characteristics of widely used PD fluids by proteomics. Of 7894 identified proteins, 3871 were regulated at least by 1 and 49 by all tested PD fluids. The latter subset was enriched for cell junction-associated proteins. The different PD fluids individually perturbed proteins commonly related to cell stress, survival, and immune function pathways. Modeling two major bioincompatibility factors of PD fluids, acidosis, and glucose degradation products (GDPs) revealed distinct effects on endothelial cell function and regulation of cellular stress responses. Proteins and pathways most strongly affected were members of the oxidative stress response. Addition of the antioxidant and cytoprotective additive, alanyl-glutamine (AlaGln), to PD fluids led to upregulation of thioredoxin reductase-1, an antioxidant protein, potentially explaining the cytoprotective effect of AlaGln. In conclusion, we mapped out the molecular response of endothelial cells to PD fluids, and provided new evidence for their specific pathomechanisms, crucial for improvement of PD therapies.

Peritoneal dialysis (PD) is one therapeutic option for patients with end-stage kidney disease (ESKD). Molecular profiling of samples from PD patients using different Omics technologies has led to the discovery of dysregulated molecular processes due to PD treatment in recent years. In particular, a number of transcriptomics (TX) datasets are currently available in the public domain in the context of PD. We set out to perform a meta-analysis of TX datasets to identify dysregulated receptor-ligand interactions in the context of PD-associated complications. We consolidated transcriptomics profiles from twelve untargeted genome-wide gene expression studies focusing on human cell cultures or samples from human PD patients. Gene set enrichment analysis was used to identify enriched biological processes. Receptor-ligand interactions were identified using data from CellPhoneDB. We identified 2591 unique differentially expressed genes in the twelve PD studies. Key enriched biological processes included angiogenesis, cell adhesion, extracellular matrix organization, and inflammatory response. We identified 70 receptor-ligand interaction pairs, with both interaction partners being dysregulated on the transcriptional level in one of the investigated tissues in the context of PD. Novel receptor-ligand interactions without prior annotation in the context of PD included BMPR2-GDF6, FZD4-WNT7B, ACKR2-CCL2, or the binding of EPGN and EREG to the EGFR, as well as the binding of SEMA6D to the receptors KDR and TYROBP. In summary, we have consolidated human transcriptomics datasets from twelve studies in the context of PD and identified sets of novel receptor-ligand pairs being dysregulated in the context of PD that warrant investigation in future functional studies.

Lithium added to peritoneal dialysis fluid protects mesothelial cells and attenuates peritoneal fibrosis and angiogenesis by reducing αB-crystallin.

Long-term clinical outcome of peritoneal dialysis (PD) depends on adequate removal of small solutes and water. The peritoneal endothelium represents the key barrier and peritoneal transport dysfunction is associated with vascular changes. Alanyl-glutamine (AlaGln) has been shown to counteract PD-induced deteriorations but the effect on vascular changes has not yet been elucidated. Using multiplexed proteomic and bioinformatic analyses we investigated the molecular mechanisms of vascular pathology in-vitro (primary human umbilical vein endothelial cells, HUVEC) and ex-vivo (arterioles of patients undergoing PD) following exposure to PD-fluid. An overlap of 1813 proteins (40%) of over 3100 proteins was identified in both sample types. PD-fluid treatment significantly altered 378 in endothelial cells and 192 in arterioles. The HUVEC proteome resembles the arteriolar proteome with expected sample specific differences of mainly immune system processes only present in arterioles and extracellular region proteins primarily found in HUVEC. AlaGln-addition to PD-fluid revealed 359 differentially abundant proteins and restored the molecular process landscape altered by PD fluid. This study provides evidence on validity and inherent limitations of studying endothelial pathomechanisms in-vitro compared to vascular ex-vivo findings. AlaGln could reduce PD-associated vasculopathy by reducing endothelial cellular damage, restoring perturbed abundances of pathologically important proteins and enriching protective processes.