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RNA sequencing of mouse glomerular endothelial cells and implications of their heterogeneity in Alport syndrome

ABSTRACT: Background: Recent transcriptomic studies of total glomerular and isolated glomerular endothelial cells (GEC) provide much needed insight into the potential associations between expression of specific genes and dysfunctional endothelium in the development of diabetic kidney disease. Similarly, many studies on Alport syndrome (AS) mice, including our own, suggest that damage to GEC can contribute to disease progression. However, the underlying cellular and molecular pathways and associated transcriptional identity of the cells remain either limited or non-existent. Methods: We used a model of endothelial-specific tdTomato reporter mouse for the feasibility of isolating the GEC from kidneys of WT and AS mice. To generate a snapshot of GEC specific transcriptional profiles and to elucidate gene expression changes in AS, we performed transcriptome-wide RNA-seq analysis of GEC from AS-TektdT mice and control WT-TektdT mice at 4-month of age. Results: We identified two subpopulations of GEC (dimtdT and brighttdT) based on the fluorescence intensity of the TektdT signal. Gene expression analysis showed vast heterogeneity in the two subpopulations, including in the expression of endothelial specific genes. In AS, dimtdT and brighttdT GEC had remarkably heterogeneous profiles in matrix associated genes (Svep1, Itgβ6), metabolic activity (ApoM, Pgc1α) and immune modulation (Apelin, Icam1) compared to WT. Additionally, we confirmed the expression data of these genes in biopsy samples from AS and FSGS patients. Conclusions: Our findings suggest the presence of two subpopulations of GEC that develop distinct responses in metabolic and inflammatory immune activity in AS. In depth understanding of the pathologic role of GEC in the progression of AS could lead to novel targets for intervention.

ORGANISM(S): Mus musculus

PROVIDER: GSE135442 | GEO | 2020/07/15

REPOSITORIES: GEO

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Project description:Purpose: MicroRNA-21 contributes to the pathogenesis of fibrogenic diseases in multiple organs including the kidney. To evaluate the therapeutic utility of antimiR-21 oligonucleotides in chronic kidney disease, we silenced miR-21 in mice that develop Alport Nephropathy due to a defect in the Col4a3 gene. The goals of this study to assess the effect of inhibiting miR-21 in the Col4a3-/- Alport Syndrome mouse model at 9 weeks of age. Methods: Col4a3-/-, Col4a3+/-, and Col4a3+/+ mice in the 129X1/SvJ genetic background were obtained. Mice received anti–miR-21 (25 mg/kg) or control anti-miR (25mg/kg) in phosphate-buffered saline (PBS) by inter-scapular subcutaneous injection twice per week. In some experiments mice received a range of doses from 12.5mg/kg once a week to 50mg/kg once a week. Anti–miR-21 is a high-affinity oligonucleotide complementary to the active site of miR-21. Mice received injections starting at 24 days (3.5 weeks) after birth and ending at 5, 7, 9 or 16 weeks after birth depending on the study objectives. Total RNA from kidney tissue was extracted as per manufacturer’s instructions (miREASY kit, Qiagen). RNA quality was assessed using BioAnalyzer (Agilent). mRNA expression profiles were determined using next-generation sequencing (NGS) on the Illumina HiSeq 2000 platform producing 50bp paired-end reads. Bowtie/TopHat suites were used to align the reads to mouse genome or transcriptome and RSEM were used to quantify gene abundances. Gene level counts were then normalized with the R/Bioconductor package limma using the voom/variance stabilization method. Results: Anti-miR-21 enhanced PPAR?/RXR activity and associated downstream signaling pathways in glomerular, tubular and interstitial cells, enhanced mitochondrial function, which reduced mitochondrial ROS production and preserved tubular cell functions. In addition, inhibition of miR-21 reduced fibrogenic and inflammatory signaling in glomerular and interstitial cells, likely as a consequence of enhanced PPAR?/RXR activity and mitochondrial function. Inhibition of miR-21 represents a novel therapeutic strategy for chronic kidney diseases including Alport Nephropathy. Whole kidney mRNA profiles of wild type and Col4a3-/- mice treated with either PBS or antimiR-21, ending at 9 weeks of age, were generated by Next Generation Sequencing in triplicate using Illumina HiSeq 2000

Project description:Purpose: MicroRNA-21 contributes to the pathogenesis of fibrogenic diseases in multiple organs including the kidney. To evaluate the therapeutic utility of antimiR-21 oligonucleotides in chronic kidney disease, we silenced miR-21 in mice that develop Alport Nephropathy due to a defect in the Col4a3 gene. The goals of this study to assess the effect of inhibiting miR-21 in the Col4a3-/- Alport Syndrome mouse model at 5.5 weeks of age. Methods: Col4a3-/-, Col4a3+/-, and Col4a3+/+ mice in the 129X1/SvJ genetic background were obtained. Mice received anti–miR-21 (25 mg/kg) or control anti-miR (25mg/kg) in phosphate-buffered saline (PBS) by inter-scapular subcutaneous injection twice per week. In some experiments mice received a range of doses from 12.5mg/kg once a week to 50mg/kg once a week. Anti–miR-21 is a high-affinity oligonucleotide complementary to the active site of miR-21. Mice received injections starting at 24 days (3.5 weeks) after birth and ending at 5, 7, 9 or 16 weeks after birth depending on the study objectives. Total RNA from kidney tissue was extracted as per manufacturerâs instructions (miREASY kit, Qiagen). RNA quality was assessed using BioAnalyzer (Agilent). mRNA expression profiles were determined using next-generation sequencing (NGS) on the Illumina HiSeq 2000 platform producing 50bp paired-end reads. Bowtie/TopHat suites were used to align the reads to mouse genome or transcriptome and RSEM were used to quantify gene abundances. Gene level counts were then normalized with the R/Bioconductor package limma using the voom/variance stabilization method. Results: Anti-miR-21 enhanced PPARÎ±/RXR activity and associated downstream signaling pathways in glomerular, tubular and interstitial cells, enhanced mitochondrial function, which reduced mitochondrial ROS production and preserved tubular cell functions. In addition, inhibition of miR-21 reduced fibrogenic and inflammatory signaling in glomerular and interstitial cells, likely as a consequence of enhanced PPARÎ±/RXR activity and mitochondrial function. Inhibition of miR-21 represents a novel therapeutic strategy for chronic kidney diseases including Alport Nephropathy. Whole kidney mRNA profiles of Col4a3+/- (triplicate) and Col4a3-/- (quadruplicates) mice treated with either PBS or antimiR-21, ending at 5.5 weeks of age, were generated by Next Generation Sequencing using Illumina HiSeq 2000

Project description:Purpose: MicroRNA-21 contributes to the pathogenesis of fibrogenic diseases in multiple organs including the kidney. To evaluate the therapeutic utility of antimiR-21 oligonucleotides in chronic kidney disease, we silenced miR-21 in mice that develop Alport Nephropathy due to a defect in the Col4a3 gene. The goals of this study to assess the effect of inhibiting miR-21 in the Col4a3-/- Alport Syndrome mouse model at 5.5 weeks of age. Methods: Col4a3-/-, Col4a3+/-, and Col4a3+/+ mice in the 129X1/SvJ genetic background were obtained. Mice received anti–miR-21 (25 mg/kg) or control anti-miR (25mg/kg) in phosphate-buffered saline (PBS) by inter-scapular subcutaneous injection twice per week. In some experiments mice received a range of doses from 12.5mg/kg once a week to 50mg/kg once a week. Anti–miR-21 is a high-affinity oligonucleotide complementary to the active site of miR-21. Mice received injections starting at 24 days (3.5 weeks) after birth and ending at 5, 7, 9 or 16 weeks after birth depending on the study objectives. Total RNA from kidney tissue was extracted as per manufacturer’s instructions (miREASY kit, Qiagen). RNA quality was assessed using BioAnalyzer (Agilent). mRNA expression profiles were determined using next-generation sequencing (NGS) on the Illumina HiSeq 2000 platform producing 50bp paired-end reads. Bowtie/TopHat suites were used to align the reads to mouse genome or transcriptome and RSEM were used to quantify gene abundances. Gene level counts were then normalized with the R/Bioconductor package limma using the voom/variance stabilization method. Results: Anti-miR-21 enhanced PPARα/RXR activity and associated downstream signaling pathways in glomerular, tubular and interstitial cells, enhanced mitochondrial function, which reduced mitochondrial ROS production and preserved tubular cell functions. In addition, inhibition of miR-21 reduced fibrogenic and inflammatory signaling in glomerular and interstitial cells, likely as a consequence of enhanced PPARα/RXR activity and mitochondrial function. Inhibition of miR-21 represents a novel therapeutic strategy for chronic kidney diseases including Alport Nephropathy.

Project description:Purpose: MicroRNA-21 contributes to the pathogenesis of fibrogenic diseases in multiple organs including the kidney. To evaluate the therapeutic utility of antimiR-21 oligonucleotides in chronic kidney disease, we silenced miR-21 in mice that develop Alport Nephropathy due to a defect in the Col4a3 gene. The goals of this study to assess the effect of inhibiting miR-21 in the Col4a3-/- Alport Syndrome mouse model at 9 weeks of age. Methods: Col4a3-/-, Col4a3+/-, and Col4a3+/+ mice in the 129X1/SvJ genetic background were obtained. Mice received anti–miR-21 (25 mg/kg) or control anti-miR (25mg/kg) in phosphate-buffered saline (PBS) by inter-scapular subcutaneous injection twice per week. In some experiments mice received a range of doses from 12.5mg/kg once a week to 50mg/kg once a week. Anti–miR-21 is a high-affinity oligonucleotide complementary to the active site of miR-21. Mice received injections starting at 24 days (3.5 weeks) after birth and ending at 5, 7, 9 or 16 weeks after birth depending on the study objectives. Total RNA from kidney tissue was extracted as per manufacturer’s instructions (miREASY kit, Qiagen). RNA quality was assessed using BioAnalyzer (Agilent). mRNA expression profiles were determined using next-generation sequencing (NGS) on the Illumina HiSeq 2000 platform producing 50bp paired-end reads. Bowtie/TopHat suites were used to align the reads to mouse genome or transcriptome and RSEM were used to quantify gene abundances. Gene level counts were then normalized with the R/Bioconductor package limma using the voom/variance stabilization method. Results: Anti-miR-21 enhanced PPARα/RXR activity and associated downstream signaling pathways in glomerular, tubular and interstitial cells, enhanced mitochondrial function, which reduced mitochondrial ROS production and preserved tubular cell functions. In addition, inhibition of miR-21 reduced fibrogenic and inflammatory signaling in glomerular and interstitial cells, likely as a consequence of enhanced PPARα/RXR activity and mitochondrial function. Inhibition of miR-21 represents a novel therapeutic strategy for chronic kidney diseases including Alport Nephropathy.

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RNA sequencing of mouse glomerular endothelial cells and implications of their heterogeneity in Alport syndrome

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