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

Grgic, Ivica; Hofmeister, Andreas; Bernhardy, Andrea J.; Sun, Hua; Maarouf, Omar H.; Bijol, Vanesa; Pollak, Martin R.; Humphreys, Benjamin D.

doi:10.1038/ki.2014.204

Cited by 42 publications

(45 citation statements)

References 63 publications

(81 reference statements)

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“…RNA-seq of FACS sorted cells 6 and translating ribosome affinity purification (TRAP) 7-9 have provided great insight into the molecular signatures and gene regulatory networks for specific cell types in kidney development, homeostasis and disease. However, these techniques require advance knowledge of cell markers to define cell types.…”

Section: Introductionmentioning

confidence: 99%

The promise of single-cell RNA sequencing for kidney disease investigation

Humphreys

2017

Kidney International

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New techniques for single cell RNA sequencing (scRNA-seq) at high throughput are leading to profound new discoveries in biology. The ability to generate vast amounts of transcriptomic data at cellular resolution represents a transformative advance allowing the identification of novel cell types, states and dynamics. In this review, we summarize the development of scRNA-seq methodologies and highlight their advantages and drawbacks. We discuss available software tools for analyzing scRNA-Seq data and summarize current computational challenges. Finally, we outline ways in which this powerful technology might be applied to discovery research in kidney development and disease.

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Section: Introductionmentioning

confidence: 99%

The promise of single-cell RNA sequencing for kidney disease investigation

Humphreys

2017

Kidney International

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“…6,7 However, due to the heterogeneity in cell types, these data provide limited information specifically on podocyte injury. Recently, mRNA profiles obtained directly from podocytes have been reported, 8,9 but such information from the diabetic kidney has not been determined.…”

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confidence: 99%

“…A recent approach of translating ribosome affinity purification which avoids the digestion and sorting processes may provide better information on mRNA profiles of podocytes. 9 Future studies are required to compare the two approaches and to determine whether the translating ribosome affinity purification method may yield more information on the mechanism of podocyte injury in DN. Third, although each podocyte sample contained pooled podocytes sorted from five mice, the number of sample size per group used for sequencing was still small.…”

mentioning

confidence: 99%

Comparison of Glomerular and Podocyte mRNA Profiles in Streptozotocin-Induced Diabetes

Wei²,

Lee³

et al. 2016

Journal of the American Society of Nephrology

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Evaluating the mRNA profile of podocytes in the diabetic kidney may indicate genes involved in the pathogenesis of diabetic nephropathy. To determine if the podocyte-specific gene information contained in mRNA profiles of the whole glomerulus of the diabetic kidney accurately reflects gene expression in the isolated podocytes, we crossed Nos3 2/2 IRG mice with podocin-rtTA and TetON-Cre mice for enhanced green fluorescent protein labeling of podocytes before diabetic injury. Diabetes was induced by streptozotocin, and mRNA profiles of isolated glomeruli and sorted podocytes from diabetic and control mice were examined 10 weeks later. Expression of podocyte-specific markers in glomeruli was downregulated in diabetic mice compared with controls. However, expression of these markers was not altered in sorted podocytes from diabetic mice. When mRNA levels of glomeruli were corrected for podocyte number per glomerulus, the differences in podocyte marker expression disappeared. Analysis of the differentially expressed genes in diabetic mice also revealed distinct upregulated pathways in the glomeruli (mitochondrial function, oxidative stress) and in podocytes (actin organization). In conclusion, our data suggest reduced expression of podocyte markers in glomeruli is a secondary effect of reduced podocyte number, thus podocyte-specific gene expression detected in the whole glomerulus may not represent that in podocytes in the diabetic kidney.

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“…We have shown previously that ACTN4 potentiates transcriptional activation by MEF2 (25,26), NF-B (27,28), and NRs (29 -31). The newly identified nuclear function of ACTN4, its link to FSGS and MCD, and the beneficial effects of GCs on FSGS and MCD prompted us to investigate the role of ACTN4 in GC-mediated transcriptional regulation in podocytes and the effects of FSGS-linked ACTN4 mutations on GR transactivation activity.…”

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confidence: 99%

α Actinin 4 (ACTN4) Regulates Glucocorticoid Receptor-mediated Transactivation and Transrepression in Podocytes

Zhao

Khurana

Charkraborty

et al. 2017

Journal of Biological Chemistry

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Edited by Xiao-Fan WangGlucocorticoids are a general class of steroids that possess renoprotective activity in glomeruli through their interaction with the glucocorticoid receptor. However, the mechanisms by which glucocorticoids ameliorate proteinuria and glomerular disease are not well understood. In this study, we demonstrated that ␣ actinin 4 (ACTN4), an actin-cross-linking protein known to coordinate cytoskeletal organization, interacts with the glucocorticoid receptor (GR) in the nucleus of human podocytes (HPCs), a key cell type in the glomerulus critical for kidney filtration function. The GR-ACTN4 complex enhances glucocorticoid response element (GRE)-driven reporter activity. Stable knockdown of ACTN4 by shRNA in HPCs significantly reduces dexamethasone-mediated induction of GR target genes and GRE-driven reporter activity without disrupting dexamethasone-induced nuclear translocation of GR. Synonymous mutations or protein expression losses in ACTN4 are associated with kidney diseases, including focal segmental glomerulosclerosis, characterized by proteinuria and podocyte injury. We found that focal segmental glomerulosclerosis-linked ACTN4 mutants lose their ability to bind liganded GR and support GRE-mediated transcriptional activity. Mechanistically, GR and ACTN4 interact in the nucleus of HPCs. Furthermore, disruption of the LXXLL nuclear receptor-interacting motif present in ACTN4 results in reduced GR interaction and dexamethasone-mediated transactivation of a GRE reporter while still maintaining its actin-binding activity. In contrast, an ACTN4 isoform, ACTN4 (Iso), that loses its actin-binding domain is still capable of potentiating a GRE reporter. Dexamethasone induces the recruitment of ACTN4 and GR to putative GREs in dexamethasone-transactivated promoters, SERPINE1, ANGPLT4, CCL20, and SAA1 as well as the NF-B (p65) binding sites on GR-transrepressed promoters such as IL-1␤, IL-6, and IL-8. Taken together, our data establish ACTN4 as a transcriptional co-regulator that modulates both dexamethasone-transactivated and -transrepressed genes in podocytes.

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

Cited by 42 publications

References 63 publications

The promise of single-cell RNA sequencing for kidney disease investigation

The promise of single-cell RNA sequencing for kidney disease investigation

Comparison of Glomerular and Podocyte mRNA Profiles in Streptozotocin-Induced Diabetes

α Actinin 4 (ACTN4) Regulates Glucocorticoid Receptor-mediated Transactivation and Transrepression in Podocytes

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