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Urine single cell RNA-sequencing in focal segmental glomerulosclerosis reveals inflammatory signatures in immune cells and podocytes

ABSTRACT: The diagnosis of focal segmental glomerulosclerosis (FSGS) requires a renal biopsy which is invasive and can be problematic in children and in some adults. We used single cell RNA-sequencing to explore disease-related cellular signatures in 17 urine samples from 12 FSGS subjects. We identified immune cells in urine predominantly monocytes and renal epithelial cells including podocytes. Analysis revealed M1 and M2 monocyte subsets and podocytes showing increased expression of genes involved in epithelial-to-mesenchymal transition (EMT). We confirmed M1 and M2 gene signatures using published monocyte/macrophage data from lupus nephritis and cancer. Using renal transcriptomic data from the Nephrotic Syndrome Study Network (NEPTUNE) we found that urine immune and EMT signature genes also showed higher expression in FSGS biopsies compared to minimal change disease biopsies. These results suggest that urine cell profiling may serve as a diagnostic and prognostic tool in nephrotic syndrome and may assist in identifying novel biomarkers and developing personalized therapeutic strategies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE176465 | GEO | 2021/12/07

REPOSITORIES: GEO

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Project description:Peritoneal dialysis (PD) is the worldwide recognized preferred dialysis treatment for children affected by end-stage kidney disease (ESKD). However, due to the unphysiological composition of PD fluids, the peritoneal membrane (PM) of these patients may undergo structural and functional alterations, which may cause fibrosis. Several factors may accelerate this process and primary kidney disease may have a causative role. In particular, patients affected by corticoresistant primary focal segmental glomerulosclerosis), a rare glomerular disease leading to nephrotic syndrome and ESKD, seem more prone to develop peritoneal fibrosis. The mechanism causing this predisposition is still unrecognized. To better define this condition, we carried out, for the first time, a new comprehensive comparative proteomic mass spectrometry analysis of mesothelial exosomes from peritoneal dialysis effluent (PDE) of 6 pediatric patients with focal segmental glomerular sclerosis (FSGS) versus 6 patients affected by other primary renal diseases (No FSGS). Our omic study demonstrated that, despite the high overlap in the protein milieu between the two study groups, machine learning allowed a complete distinction of the whole proteomic exosome mesothelial content of FSGS versus No FSGS (with 100% accuracy). Out of the 2490 identified proteins, 40% (995) were involved in epithelial-mesenchymal transition (EMT)/fibrosis and in the transforming growth factor-β pathway. Additionally, the Weight Gene Co-expression Network Analysis algorithm identified that some of the discriminative proteins (TIMP1, CTHRC1, SPARC, CHMP4B, COL5A2, ANXA13, FNC2 and CENP-E) were also highly correlated to PD vintage, fibrosis, EMT and non-neoplastic PM disease. All together our data demonstrated that mesothelial cells of FSGS patients are more prone to activate a pro-fibrotic machinery with exosomes having a primary role in this process. Moreover, they indicated that identified FSGS-associated elements in mesothelial exosome protein could be employed as potential new biomarkers of mesothelial integrity. Finally, our results highlighted that in FSGS patients particular attention should be paid to use more biocompatible dialysis solution, reduce the length of time on PD and personalize PD regimens to minimize the risk of rapid loss of PM function or development of encapsulating peritoneal sclerosis.

Project description:Focal and segmental glomerulosclerosis (FSGS) is a severe form of idiopathic nephrotic syn-drome (INS), a glomerulopathy of presumable immune origin and attributed to extrarenal path-ogenic circulating factors. FSGS recurrence (rFSGS) after transplant occurs in 30 to 50% of cases. Direct analysis of patient plasma proteome has been scarcely addressed to date, mainly due to methodological difficulties associated with plasma complexity and dynamic range. In this study, firstly we compared different methods of plasma preparation, secondly we compared the plasma proteome of rFSGS and controls using two preparation methods, and thirdly we analyzed the early proximal signaling events in podocytes subjected to patient plasma, by a combination of phosphoproteomics and lipid raft proteomics (raftomics). By combining immunodepletion and high pH fractionation, we performed a differential proteomic analysis of soluble plasma pro-teins and of extracellular vesicles (EV) obtained from healthy controls, non-INS patient controls, and rFSGS patients (n=4). In both soluble and EV protein sets from rFSGS patients we found a statistically significant increase in a cluster of proteins involved in neutrophil degranulation. A group of lipid binding proteins, generally associated with lipoproteins, was found decreased in the soluble set from rFSGS patients. In addition, three aminoacid transporters involved in mTORC1 activation were found significantly increased in EV from rFSGS. Next, we incubated human podocytes for 30min with 10% plasma from both groups of patients. Phosphoproteomics and raftomics of podocytes revealed profound differences in proteins involved in the mTOR pathway, in autophagy, and in cytoskeleton organization. We analyzed the correlation between the abundance of plasma and plasma-regulated podocyte proteins. The observed changes high-light some of the mechanisms involved in FSGS recurrence and could be used as specific early markers of circulating factor activity on podocytes.

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Urine single cell RNA-sequencing in focal segmental glomerulosclerosis reveals inflammatory signatures in immune cells and podocytes

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