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

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

PROVIDER: PRJNA736336 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR14766600.fastq.gz	Fastqsanger.gz
	SRR14766601.fastq.gz	Fastqsanger.gz
	SRR14766602.fastq.gz	Fastqsanger.gz
	SRR14766603.fastq.gz	Fastqsanger.gz
	SRR14766604.fastq.gz	Fastqsanger.gz

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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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