A novel method for isolating podocytes using magnetic activated cell sorting

Murakami, Ayumi; Oshiro, Hisashi; Kanzaki, Seiichi; Yamaguchi, Akira; Yamanaka, Shōji; Furuya, Mitsuko; Miura, Satoshi; Kanno, Hiroshi; Nagashima, Yoji; Aoki, Ichiro; Nagahama, Kiyotaka

doi:10.1093/ndt/gfq323

Cited by 28 publications

(22 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Dynabead perfusion combined with enzymatic digestion and sieving methods has proven effective in isolating glomeruli from mouse kidney at a large scale in vivo and has greatly facilitated the establishment of glomerular transcription profiles (6–8). Very recently, magnetic bead and transgenic models for FACS purification have been successfully applied to separate podocytes from other glomerular cells improving enrichment of podocyte specific RNA (9–11). These techniques however require enzymatic and mechanical disaggregation of kidney tissue for the creation of single cell suspensions that introduce a stress-response gene signature.…”

Section: Introductionmentioning

confidence: 99%

Discovery of new glomerular disease–relevant genes by translational profiling of podocytes in vivo

Grgic

Hofmeister

Bernhardy

et al. 2014

Kidney International

View full text Add to dashboard Cite

Identifying new biomarkers and therapeutic targets for podocytopathies such as focal segmental glomerulosclerosis (FSGS) requires a detailed analysis of transcriptional changes in podocytes over the course of disease. Here we used translating ribosome affinity purification (TRAP) to isolate and profile podocyte-specific mRNA in two different models of FSGS. Expressed eGFP-tagged ribosomal protein L10a in podocytes under the control of the Collagen-1α1 promoter enabled podocyte-specific mRNA isolation in a one-step process over the course of disease. This TRAP protocol robustly enriched known podocyte-specific mRNAs. We crossed col1α1-L10a mice with the actn4−/− and actn4+/K256E models of FSGS and analyzed podocyte transcriptional profiles at 2, 6 and 44 weeks of age. Two upregulated podocyte genes in murine FSGS (CXCL1 and DMPK) were found to be upregulated at the protein level in biopsies from patients with FSGS, validating this approach. There was no dilution of podocyte-specific transcripts during disease. These are the first podocyte-specific RNA expression datasets during aging and in two models of FSGS. This approach identified new podocyte proteins that are upregulated in FSGS and help define novel biomarkers and therapeutic targets for human glomerular disease.

show abstract

Section: Introductionmentioning

confidence: 99%

Discovery of new glomerular disease–relevant genes by translational profiling of podocytes in vivo

Grgic

Hofmeister

Bernhardy

et al. 2014

Kidney International

View full text Add to dashboard Cite

show abstract

“…Immortalized human renal tubular epithelial cells (HK-2, ATCC) and HEK293 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS, Hyclone). Primary tubular epithelial cells (Xu et al, 2015), primary podocytes (Murakami et al, 2010), and primary renal fibroblast cells (Kunzel et al, 2019)were isolated as previously described. HDAC11 expression plasmids (Watanabe et al, 2014), HDAC11 promoter-luciferase constructs (Voelter-Mahlknecht et al, 2005), and KLF15 promoter-luciferase constructs (Shao et al, 2018) have been previously described.…”

Section: Cell Culture Plasmids Transient Transfection and Reportermentioning

confidence: 99%

Histone Deacetylase 11 Contributes to Renal Fibrosis by Repressing KLF15 Transcription

Mao

Liu

Zhang

et al. 2020

Front. Cell Dev. Biol.

View full text Add to dashboard Cite

Renal fibrosis represents a key pathophysiological process in patients with chronic kidney diseases (CKD) and is typically associated with a poor prognosis. Renal tubular epithelial cells (RTECs), in response to a host of pro-fibrogenic stimuli, can trans-differentiate into myofibroblast-like cells and produce extracellular matrix proteins to promote renal fibrosis. In the present study we investigated the role of histone deacetylase 11 (HDAC11) in this process and the underlying mechanism. We report that expression levels of HDAC11 were up-regulated in the kidneys in several different animal models of renal fibrosis. HDAC11 was also up-regulated by treatment of Angiotensin II (Ang II) in cultured RTECs. Consistently, pharmaceutical inhibition with a smallmolecule inhibitor of HDAC11 (quisinostat) attenuated unilateral ureteral obstruction (UUO) induced renal fibrosis in mice. Similarly, HDAC11 inhibition by quisinostat or HDAC11 depletion by siRNA blocked Ang II induced pro-fibrogenic response in cultured RTECs. Mechanistically, HDAC11 interacted with activator protein 2 (AP-2α) to repress the transcription of Kruppel-like factor 15 (KLF15). In accordance, KLF15 knockdown antagonized the effect of HDAC11 inhibition or depletion and enabled Ang II to promote fibrogenesis in RTECs. Therefore, we data unveil a novel AP-2α-HDAC11-KLF15 axis that contributes to renal fibrosis.

show abstract

“…Podocytes were isolated as previously described (Murakami et al, 2010). Briefly, following endothelial cell depletion using the anti-CD31 antibody (BL102503; BioLegend), podocytes were isolated from minced murine kidneys using magnet-activated cell sorting with an anti-nephrin antibody (sc-19000; Santa Cruz Biotechnology).…”

Section: Podocyte Isolationmentioning

confidence: 99%

Decreased KAT5 Expression Impairs DNA Repair and Induces Altered DNA Methylation in Kidney Podocytes

Hishikawa

Hayashi

Abe³

et al. 2019

Cell Reports

View full text Add to dashboard Cite

Graphical Abstract Highlights d KAT5-mediated DNA damage repair is essential for podocyte maintenance d KAT5 is decreased in diabetic nephropathy podocytes d Impaired DNA damage repair may be associated with altered DNA methylation status d KAT5 may be a therapeutic target for diabetic nephropathy SUMMARY Altered DNA methylation plays an important role in the onset and progression of kidney disease. However, little is known about how the changes arise in disease states. Here, we report that KAT5-mediated DNA damage repair is essential for the maintenance of kidney podocytes and is associated with DNA methylation status. Podocyte-specific KAT5-knockout mice develop severe albuminuria with increased DNA double-strand breaks (DSBs), increased DNA methylation of the nephrin promoter region, and decreased nephrin expression. Podocyte KAT5 expression is decreased, whereas DNA DSBs and DNA methylation are increased in diabetic nephropathy; moreover, KAT5 restoration by gene transfer attenuates albuminuria. Furthermore, KAT5 decreases DNA DSBs and DNA methylation at the same nephrin promoter region, which indicates that KAT5-mediated DNA repair may be related to DNA methylation status. These results suggest a concept in which an environment of DNA damage repair, which occurs with decreased KAT5, may affect DNA methylation status.

show abstract

A novel method for isolating podocytes using magnetic activated cell sorting

Cited by 28 publications

References 31 publications

Discovery of new glomerular disease–relevant genes by translational profiling of podocytes in vivo

Discovery of new glomerular disease–relevant genes by translational profiling of podocytes in vivo

Histone Deacetylase 11 Contributes to Renal Fibrosis by Repressing KLF15 Transcription

Decreased KAT5 Expression Impairs DNA Repair and Induces Altered DNA Methylation in Kidney Podocytes

Contact Info

Product

Resources

About