Project description:Investigation of DNAse Hypersensitivity changes in podocytes cultured under normal or high glucose conditions transfected with a miR-93 mimic or a nontargeting mimic.
Project description:Investigation of mRNA changes in podocytes transfected with a miR-93 mimic or a nontargeting mimic. The design was meant to identify biologically significant, novel targets of the miR-93 microRNA in podocytes
Project description:Investigation of DNAse Hypersensitivity changes in podocytes cultured under normal or high glucose conditions transfected with a miR-93 mimic or a nontargeting mimic. Examination of the changes in hypersensitivity induced by high glucose culture conditions compared to normal glucose conditions to mimic the diabetic millieu. Further, to see if miR-93 overexpression can reverse these changes.
Project description:Transcriptomes of differentiated cells of the conditionally immortalized mouse podocyte cell line SVI (Schiwek et al., Kidney Int. 66: 91-101, 2004) were determined as described in Warsow et al. (Kidney Int. 84: 104-115, 2013) after application of mechanical stress (Endlich et al., J. Am. Soc. Nephrol. 12: 413-422, 2001) as compared to control conditions. Elevated glomerular pressure represents a high risk for the development of severe kidney diseases and causes an increase of mechanical load to podocytes. In this study we investigated whether mechanical stress alters gene expression in cultured podocytes using gene arrays. We found that tetraspanin CD9 is significantly upregulated in cultured podocytes after mechanical stress. The differential expression of CD9 was confirmed by RT-PCR and Western blot under stretched and unstretched conditions. Furthermore, mechanical stress resulted in a relocalization of CD9. To get an insight into the functional role of CD9, podocytes were transfected with pEGFP-CD9. The expression of CD9 induced the formation of substratum-attached thin arborized protrusions (TAPs). Ca2+ depletion revealed that podocytes over-expressing CD9 possess altered adhesive properties in contrast to the control transfected cells. Finally, elevated CD9 expression increased migration of podocytes in a wound assay. In summary, our results suggest that upregulation of CD9 may play an important role in podocyte morphology, adhesion and migration.
Project description:Transcriptomes of differentiated cells of the conditionally immortalized mouse podocyte cell line SVI (Schiwek et al., Kidney Int. 66: 91-101, 2004) were determined as described in Warsow et al. (Kidney Int. 84: 104-115, 2013) after application of mechanical stress (Endlich et al., J. Am. Soc. Nephrol. 12: 413-422, 2001) as compared to control conditions. Elevated glomerular pressure represents a high risk for the development of severe kidney diseases and causes an increase of mechanical load to podocytes. In this study we investigated whether mechanical stress alters gene expression in cultured podocytes using gene arrays. We found that tetraspanin CD9 is significantly upregulated in cultured podocytes after mechanical stress. The differential expression of CD9 was confirmed by RT-PCR and Western blot under stretched and unstretched conditions. Furthermore, mechanical stress resulted in a relocalization of CD9. To get an insight into the functional role of CD9, podocytes were transfected with pEGFP-CD9. The expression of CD9 induced the formation of substratum-attached thin arborized protrusions (TAPs). Ca2+ depletion revealed that podocytes over-expressing CD9 possess altered adhesive properties in contrast to the control transfected cells. Finally, elevated CD9 expression increased migration of podocytes in a wound assay. In summary, our results suggest that upregulation of CD9 may play an important role in podocyte morphology, adhesion and migration. Three independent batches were used.
Project description:Purpose: Next-generation sequencing (NGS) was used to define the transcriptome of native mouse podocytes and non-podocytes glomerular cells as part of a project aiming to define the molecular fingerprint of mouse podocytes. Method: Glomeruli from 29 Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J x hNPHS2Cre mice at the age of 10 weeks were purified and a single cell solution was prepared to seperate GFP-expressing (podocytes) and GFP-negative (non-podocytes glomerular cells) cells by FACS sorting. RNA was extracted and prepared for further analysis using directional, polyA+ library preparation. An Illumina HiSeq2500 was used for a paired-end sequencing of 100 cycles . Salmon and Sleuth were used for downstream analysis. Results: A total of 100 Million reads each from podocytes and non-podocytes glomerular cells could be used for further analysis.
Project description:Genome wide DNA methylation profiling of control (vector-transfected) and KLF4-overexpressing human podocytes (parental cell line: Saleem et al. JASN 13:630, 2002). The Illumina HumanMethylation450 Beadchip was used to obtain DNA methylation profiles. Bisulphite converted DNA samples from control (vector-transfected, n=1) and KLF4-overexpressing human podocytes (n=1) were hybridised to the Illumina HumanMethylation450 Beadchip and DNA methylation was compared.
Project description:We generated a genome wide map of instances where the long noncoding RNA, Tug1, binds to DNA in cultured mouse podocytes under normal glucose conditions using Chromatin-RNA Precipitation coupled with high throughput sequencing (ChIRP-Seq) 48 alternating (even, odd) biotynilated probes were designed to span the full length of Tug1 RNA. Chromatin was prepared from gluteraldehyde crosslinked nuclei from early passage podocytes. Chromatin extracts were duplicated with either even or odd probes. Duplicate samples for Input DNA, Even pulldown (PD) and Odd PD DNA was purified following incubation and supplied for Illumina sequencing by ArrayStar (Rockville, MD).
Project description:Genome wide DNA methylation profiling of control (vector-transfected) and KLF4-overexpressing human podocytes (parental cell line: Saleem et al. JASN 13:630, 2002). The Illumina HumanMethylation450 Beadchip was used to obtain DNA methylation profiles.