RNA-seq of cultured human kidney peritubular microvascular endothelial cells following exposure to cyclosporine A
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ABSTRACT:
PROVIDER: PRJNA429146 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR6451420_1.fastq.gz | Fastqsanger.gz | |||
| SRR6451420_2.fastq.gz | Fastqsanger.gz | |||
| SRR6451421_1.fastq.gz | Fastqsanger.gz | |||
| SRR6451421_2.fastq.gz | Fastqsanger.gz | |||
| SRR6451422_1.fastq.gz | Fastqsanger.gz |
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Project description:This study represents the first time human kidney endothelial cells were exposed to cyclosporine A (CsA), a calcineurin inhibitor known to contribute to nephrotoxicity with symptoms including microvascular injury. This toxicity has been found to be unique to human trials and human kidneys are particularly susceptible to injury. We found that human kidney peritubular microvascular endothelilal cells (HKMECs) that were exposed to CsA exhibited transcriptomic changes around genes important to VEGF signaling and endothelial inflammation.
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Project description:Endothelial cells in the blood vessels in the kidney exert different functions depending on the (micro)vascular bed they are located in. These functional differences are likely a result of differential microRNA and mRNA transcription patterns, yet the identity of these molecules is not well known. We zoomed in on the endothelial cells of microvascular compartments in mouse renal cortex by laser microdissecting the microvessels prior to (small) RNA sequencing analyses. By these means, we characterised microRNA and mRNA transcription profiles of arterioles, glomeruli, peritubular capillaries, and post-capillary venules. RT-qPCR, in situ hybridisation, and immunohistochemistry were used to validate sequencing results. Unique microRNA and mRNA transcription profiles were found in all microvascular compartments, with dedicated marker microRNAs and mRNAs showing enriched transcription in a single microvascular compartment. In situ hybridisation validated localisation of microRNAs mmu-miR-140-3p in arterioles, of mmu-miR-322-3p in glomeruli, and of mmu-miR-451a in post-capillary venules. Immunohistochemical staining showed that von Willebrand Factor protein was mainly expressed in arterioles and post-capillary venules, while GABRB1 expression was enriched in glomeruli and IGF1 in post-capillary venules. Our study shows that microvascular endothelial heterogeneity in mouse kidney is a result of a combination of differentially expressed microRNAs and mRNAs. The identified profiles provide important molecular information to take into account for future studies into microvascular engagement in health and disease.
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Project description:Endothelial cells in the blood vessels in the kidney exert different functions depending on the (micro)vascular bed they are located in. These functional differences are likely a result of differential microRNA and mRNA transcription patterns, yet the identity of these molecules is not well known. We zoomed in on the endothelial cells of microvascular compartments in mouse renal cortex by laser microdissecting the microvessels prior to (small) RNA sequencing analyses. By these means, we characterised microRNA and mRNA transcription profiles of arterioles, glomeruli, peritubular capillaries, and post-capillary venules. RT-qPCR, in situ hybridisation, and immunohistochemistry were used to validate sequencing results. Unique microRNA and mRNA transcription profiles were found in all microvascular compartments, with dedicated marker microRNAs and mRNAs showing enriched transcription in a single microvascular compartment. In situ hybridisation validated localisation of microRNAs mmu-miR-140-3p in arterioles, of mmu-miR-322-3p in glomeruli, and of mmu-miR-451a in post-capillary venules. Immunohistochemical staining showed that von Willebrand Factor protein was mainly expressed in arterioles and post-capillary venules, while GABRB1 expression was enriched in glomeruli and IGF1 in post-capillary venules. Our study shows that microvascular endothelial heterogeneity in mouse kidney is a result of a combination of differentially expressed microRNAs and mRNAs. The identified profiles provide important molecular information to take into account for future studies into microvascular engagement in health and disease.
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Project description:To elucidate the gene profile of anti-fibroproliferative effects of cyclosporine, we added TGF-b and/or CsA to MRC5 (fetal human lung fibroblast) cell line. After 24 hours serum starvation, fibroblasts were treated with 3 ng/ml TGF-β1 and were treated with or without 2μg/ml Cyclosporine and effects were examined at 48 hours after treatment. Expression of three genes (IGFBP3, ID1 and PPARG) from this signature was quantified in the same RNA samples by real-time PCR. TGF-β and cyclosporine induced gene expression data was measured in MRC5 (fetal human fibroblast cell line) at 48h after treatments. The dose of TGF-β was 3 ng/ml and cyclosporine was 2 μg/ml. The comparison was done between four groups (control, TGF-β, cyclosporine and TGF-β plus cyclosporine).Three independent experiments were performed at each treatments using different samples for each experiment.
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Project description:Kidney transplant recipients with biopsy-proven microvascular injury (MVI) have increased risk for allograft failure. MVI is often caused by antibody-mediated injury that is resistant to available treatments. Current diagnostic methods are also inadequate, with interobserver variability in traditional pathology reads, variable assessment of circulating donor-specific antibody between HLA laboratories, and peritubular capillary C4d staining. Molecular assessments of kidney biopsies can provide improved sensitivity for diagnosing MVI and other allograft pathology, while improving reproducibility and objectivity. Most molecular classifiers have been based on whole genome sequencing to develop diagnostic tests, but have provided limited therapeutic targets. In this study, we pursued a candidate gene approach to measure WNT pathway genes in residual clinical FFPE biopsies with and without MVI. We focused on the WNT pathway because of previous translational studies that implicated this pathway in chronic renal allograft injury as well as vascular injury in native chronic kidney disease.
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Project description:Kidney transplant recipients with biopsy-proven microvascular injury (MVI) have increased risk for allograft failure. MVI is often caused by antibody-mediated injury that is resistant to available treatments. Current diagnostic methods are also inadequate, with interobserver variability in traditional pathology reads, variable assessment of circulating donor-specific antibody between HLA laboratories, and peritubular capillary C4d staining. Molecular assessments of kidney biopsies can provide improved sensitivity for diagnosing MVI and other allograft pathology, while improving reproducibility and objectivity. Most molecular classifiers have been based on whole genome sequencing to develop diagnostic tests, but have provided limited therapeutic targets. In this study, we pursued a candidate gene approach to measure WNT pathway genes in residual clinical FFPE biopsies with and without MVI. We focused on the WNT pathway because of previous translational studies that implicated this pathway in chronic renal allograft injury as well as vascular injury in native chronic kidney disease. Case-control study of 95 residual FFPE biopsies with MVI (g+ptc score >= 2, n=50) or Stable (g+ptc score < 2 and no other major abnormalities, n=45). Biopsies were retrieved from a biorepository of over 500 kidney transplant biopsies. We compared expression of 180 WNT pathway genes and 30 custom skipe-in targets (derived from previous studies of endothelial injury in transplantation) between MVI and Stable groups, with correction for multiple comparisons using FDR < 5%. This dataset is part of the TransQST collection.
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