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Minimal change disease (MCD) is the most common form of idiopathic nephrotic syndrome in children, usually responsive to corticosteroid therapy. Both the etiology and the pathogenesis remain elusive. Identification of genes differentially expressed by certain kidney cells and other cell populations in healthy individuals and patients with MCD may help to elucidate the mechanism of MCD and hence identify targets for drug development. One of the major limitations in this approach is the fact that kidney biopsy is only rarely performed in patients with steroid-sensitive nephrotic syndrome. Moreover, animal models, intensively developed in recent decades, only partially re-capitulate the natural course of human MCD.
We use urinary cell sediment (U-sed) as a representative population of cells relevant to MCD, the only non-invasive source of wide spectrum of cells located in patient’s kidney and urinary tract. Traditional single-cell RNA sequencing (scRNA-seq) methods are restricted to the use of fresh, viable cells and are limited to an in-house core facility. This makes collecting samples from healthy subjects and MCD patients at various stages of the disease (during relapse and remission [on- or off- immuno-suppressants]) mostly challenging. In this study, we compare two new scRNA-seq platforms that allow to separate single cells immediately after sample collection and quality control, and store them over time. It is noteworthy that this procedure can be performed in any non-specialized laboratory. Library construction, which involves synthesizing first-strand cDNA from captured mRNA transcripts and attaching barcodes unique to each cell, can be performed later. Subsequently, the libraries are sequenced.
Preliminary results confirm that U-sed is a suitable tool to study resident glomerular cells, immune cells and other types of cells located adjacent to the podocytes, which obviate the need to perform biopsies which are ethically unjustified. Differences in transcriptome in samples of urine cell sediment derived from nephrotic children and thje respective controls will follow.
U-sed scRNA-seq is a suitable tool to study glomerular processes as exemplified in MCD. Novel scRNA-seq platforms can be utilized to overcome the hurdle of analyzing fresh urine samples and allow pooling of urine samples.