Project description:This study aimed to shed light on the potential pathophysiology of MCD by using glomerular proteomic analysis. Shotgun proteomics using label-free quantitative mass spectrometry was performed on formalin-fixed, paraffin-embedded (FFPE) renal biopsies from two groups of samples: control (CTR) and MCD. Glomeruli were excised from FFPE renal biopsies using laser capture microdissection (LCM), and a single-pot solid-phase-enhanced sample preparation (SP3) digest method was used to improve yield and protein identifications.
Project description:Urine passes through the entire kidney and urinary tract system starting from the glomerulus and ending to the urethra. Cells in the kidney and urinary tract could be exfoliated from the epithelium into the urine, while leukocyte could infiltrate from the local tissue into the urine, which makes the urine a useful subject for clinical evaluation of relevant diseases. We performed scRNA-seq on voided urine samples. 50–100 mL middle stream urine samples were collected from 12 Chinese healthy adults and combined for droplet-based single-cell RNA sequencing after flow cytometric sorting of live cells. We presented the first single-cell atlas of adult human urine and identified multiple previously unrecognized cell types. Based on our scRNA-seq analysis data, a SOX9+ cell population was identified in adult human urine which we speculated to have progenitor potential.
Project description:Early diagnosis and treatment is pivotal to the management of kidney disease, whereas the pathological mechanisms and minimally invasive diagnostic method still need to be investigated. In the present study, single-cell RNA sequencing (scRNA-seq) was used to evaluate the heterogeneity of kidney diseases in single cell level. Cellular gene expression profiles of cells of renal tissue, peripheral blood mononuclear cells (PBMCs) and urine from four nephritis patients were performed. Our analysis revealed 12 subsets of renal cells and leukocytes, including fibroblast cells, mesangial cells, epithelial cells, proximal tubule cells (PTCs), and 6 types of immune cells, CD8+ T cell, macrophages (MC), nature killer cells (NK), dendritic cells (DC), B cell and neutrophils. PTCs were detected in both PBMC and urine, while PTC was negative in healthy blood sample. Multiple populations of fibroblast cells, mesangial cells and PTCs demonstrated pro-inflammatory or pro-apoptotic responses. Gene expression analysis suggested that chemotactic and activating effect of inflammatory PTCs and fibroblasts on neutropils were critical for the development and progress of nephritis, which was supported by the widely overexpressed pro-inflammatory genes in these cells. Gene expression profiles of inflammatory PTCs in PBMC, urine and kidney are highly correlated, indicating the high possibility of urine and PBMC PTCs in serving as a surrogate for kidney biopsies.
Project description:Background: Urine is a potential source of biomarkers for diseases of the kidneys and urinary tract. RNA, including microRNA, is present in the urine enclosed in detached cells or in extracellular vesicles (EVs) or bound and protected by extracellular proteins. Detection of cell- and disease-specific microRNA in urine may aid early diagnosis of organ-specific pathology. In this study, we applied barcoded deep sequencing to profile microRNAs in urine of healthy volunteers, and characterized the effects of sex, urine fraction (cells vs. EVs) and repeated voids by the same individuals. Results: Compared to urine-cell-derived small RNA libraries, urine-EV-derived libraries were relatively enriched with miRNA, and accordingly had lesser content of other small RNA such as rRNA, tRNA and sn/snoRNA. Unsupervised clustering of specimens in relation to miRNA expression levels showed prominent bundling by specimen type (urine cells or EVs) and by sex, as well as a tendency of repeated (first and second void) samples to neighbor closely. Likewise, miRNA profile correlations between void repeats, as well as fraction counterparts (cells and EVs from the same specimen) were distinctly higher than correlations between miRNA profiles overall. Differential miRNA expression by sex was similar in cells and EVs. Conclusions: miRNA profiling of both urine EVs and sediment cells can convey biologically important differences between individuals. However, to be useful as urine biomarkers, careful consideration is needed for biofluid fractionation and sex-specific analysis, while the time of voiding appears to be less important.
Project description:Introduction: Minimal change disease (MCD) is a major cause of nephrotic syndrome. With a substantial number of patients requiring long-term immunosuppression leading to significant morbidity, the study aim was to determine MCD glomerular transcriptome to serve as a basis for biomarker discovery and novel drug target identification. Animal work showed podocyte injury induced by IL-7/IL-7R signaling (Zhai S, BBRC, 2018). Methods: Renal biopsies from adult patients representing the following groups were selected from the Norwegian Kidney Biopsy Registry: MCD (n=14), as well as normal tissue (n=8) and primary membranous nephropathy (MN; n=12) as the two reference groups. RNA for 75 base-pair paired-end RNASeq was obtained by dissecting glomeruli via laser capture microdissection (LCM) from FFPE cross-sections. Systematic delineation of condition-specific alteration in transcriptional landscapes was achieved by combining pathway-centered analyses with methodologies derived from network science and integrating multiple bioinformatics resources. Results: Compared to normal glomeruli, glomeuli from MCD displayed an inflammatory signature that appeared to be predominantly governed by the IL1 and IL7 systems. While enrichment of IL1 production and secretion was a shared feature of MCD and MN compared to normal tissue, responses involving IL7 pathway activation were unique to MCD. Indeed, IL7R expressed by glomeruli was the most up-regulated gene of to the interleukin-family in MCD vs normal controls. IL7 pathway activation was paralleled by significant enrichment in adaptive immune system processes and transcriptional regulation and depletion in pathways related to energy metabolism and transcription. Downregulation of these organ function-related themes again occurred predominately in MDC and were significantly less pronounced in MN. Conclusion: Our results demonstrate that archival FFPE-biopsies can be used to generate glomeruli-specific gene expression profiles suitable for systematic delineation of kidney-associated diseases. Here the latter provides a data-driven rationale to experimentally address these MCD-specific features as biomarkers and as novel drug targets. In this context inhibiting activation of the IL7 pathway may be particularly promising.
Project description:MicroRNA (miRNA) biomarkers for fragile X syndrome were searched by urine microRNA (miRNA) profiling using deep sequencing. The urine miRNA profile of twin boys who shared the same environment but one had a FXS full mutation and the other carried a premutation allele was compared based on the similar sequence reads. The urine of twin boys showed 28 differentiatially regulated miRNAs when 219 reliable identified miRNAs were compared.