The Use of High-Throughput Transcriptomics to Identify Pathways with Therapeutic Significance in Podocytes

Solanki, Ashish K.; Srivastava, Pankaj; Rahman, Bushra; Lipschutz, Joshua H.; Nihalani, Deepak; Arif, Ehtesham

doi:10.3390/ijms21010274

Cited by 10 publications

(10 citation statements)

References 34 publications

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“…Consistent with this analysis, E11 cells with miR-217-5p overexpression exhibited shrunken cell shapes with shortened or disorganized actin cytoskeletons. Previously, Solanki et al demonstrated that the apoptosis-related p53 pathway is involved in actin cytoskeleton damage in PAN-or ADR-treated podocytes [36]. However, in our study, the p53 pathway was not associated with predicted targets of miR-217-5p in PAN-treated podocytes.…”

Section: Discussioncontrasting

confidence: 88%

“…This possibility was supported by analyses of publicly available datasets of podocyte injury-related transcriptomes. The analysis of the dataset GSE124622, created through microarray analysis of immortalized human podocytes treated with PAN and Adriamycin (ADR) to cause podocyte injury [36], showed that Myo1d mRNA levels in the PAN-and ADR-treated podocytes decreased 0.41-fold (p = 7.54E-06) and 0.64-fold (p = 0.0364) compared to control podocytes, respectively (Table S5). Furthermore, analysis of the dataset GSE108629 analysis, created through a microarray-based study using a mouse model of immunotoxin-inducible podocyte injury, showed that Myo1d mRNA levels were reduced 0.61-fold (p = 0.00158) after podocyte injury [37].…”

Section: Gene Ontologies Associated With the Predicted Targets Of The...mentioning

confidence: 99%

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The Role of miR-217-5p in the Puromycin Aminonucleoside-Induced Morphological Change of Podocytes

Ishibashi¹,

Hayashi²,

Horikawa³

et al. 2022

ncRNA

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Podocytes, alternatively called glomerular epithelial cells, are terminally differentiated cells that wrap around glomerular capillaries and function as a part of the glomerular filtration barrier in the kidney. Therefore, podocyte injury with morphological alteration and detachment from glomerular capillaries leads to severe proteinuria and subsequent renal failure through glomerulosclerosis. Previous RNA sequencing analysis of primary rat podocytes exposed to puromycin aminonucleoside (PAN), a well-known experimental model of injured podocytes, identified several transcripts as being aberrantly expressed. However, how the expression of these transcripts is regulated remains unclear. MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally inhibit the expression of their target transcripts. In this study, using small RNA sequencing analysis, miR-217-5p was identified as the most upregulated transcript in PAN-treated rat podocytes. MiR-217-5p overexpression in E11 podocyte cells led to shrunken cells with abnormal actin cytoskeletons. Consistent with these changes in cell morphology, gene ontology (GO) enrichment analysis showed that interactive GO terms related to cell morphogenesis were enriched with the predicted targets of miR-217-5p. Of the predicted targets highly downregulated by PAN, Myosin 1d (Myo1d) is a nonmuscle myosin predicted to be involved in actin filament organization and thought to play a role in podocyte morphogenesis and injury. We demonstrated that miR-217-5p targets Myo1d by luciferase assays, qRT–PCR, and Western blotting. Furthermore, we showed that miR-217-5p was present in urine from PAN- but not saline-administrated rats. Taken together, our data suggest that miR-217-5p may serve as a therapeutic target and a biomarker for podocyte injury.

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Section: Discussioncontrasting

confidence: 88%

Section: Gene Ontologies Associated With the Predicted Targets Of The...mentioning

confidence: 99%

The Role of miR-217-5p in the Puromycin Aminonucleoside-Induced Morphological Change of Podocytes

Ishibashi¹,

Hayashi²,

Horikawa³

et al. 2022

ncRNA

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“…PPARγ binds to its promoter response elements (PPRE) along with other transcription factors such as retinoid X receptor (RXR) or to other DNA elements in association with factors such as nuclear factor kappa B (NFκB), activator protein 1 (AP1), and in proximity to CCAAT enhancer-binding proteins (C/EBPα) [5, 24, 38]. We carried out promoter element prediction using a PPARgene database (ppargene.org) [43] and cross-referenced it with bulk RNASeq GEO datasets obtained from control podocytes (GSE124622) [41] and adipocytes (GSE129153) [42]. We found that while 1669 PPRE-containing genes were detectable in both podocyte and adipocyte datasets, 453 were found to be unique to podocytes and 77 unique to adipocytes (Figure 3Ai, Supplementary Table S1).…”

Section: Resultsmentioning

confidence: 99%

Alternatively Spliced Landscape of PPARγ mRNA in Podocytes is Distinct from Adipose Tissue

Bryant

Webb

Banks

et al. 2022

Preprint

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Podocytes are highly differentiated epithelial cells, and their structural and functional integrity is compromised in a majority of glomerular and renal diseases, leading to proteinuria, chronic kidney disease, and kidney failure. Traditional agonists (e.g. pioglitazone) and selective modulators (e.g. GQ-16) of peroxisome-proliferator-activated-receptor-γ (PPARγ) reduce proteinuria in animal models of glomerular disease, and protect podocytes from injury via PPARγ activation. This indicates a pivotal role for PPARγ in maintaining glomerular function through preservation of podocytes distinct from its well-understood role in driving insulin sensitivity and adipogenesis. While its transcriptional role in activating adipokines and adipogenic genes is well-established in adipose tissue, liver and muscle, understanding of podocyte PPARγ signaling remains limited. We performed a comprehensive analysis of PPARγ mRNA variants due to alternative splicing, in human podocytes and compared with adipose tissue. We found that podocytes exclusively express the ubiquitous PPARγ Var 1 (encoding γ1) and not Var2 (encoding γ2), which is mostly restricted to adipose tissue and liver. Additionally, we detected expression at very low level of Var4, and barely detectable levels of other variants, Var3, Var11, VartORF4 and Var9, in podocytes. Podocyte vs adipose tissue expression of genes containing PPAR-promoter-response-elements suggested differential regulation by podocyte-specific PPARγ1 variant, distinct from the adipocyte-specific γ2 variant. This is also evident from distinct ingenuity-pathway-profile of RNAseq data of the PPARγ agonist-treated podocytes/glomeruli vs adipocytes/adipose tissue. In summary, podocytes and glomeruli express several PPARγ variants, including Var1 (γ1) and excluding adipocyte-specific Var2 (γ2), which may have implications in podocyte-specific signaling and pathophysiology.

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“…Since injury to podocytes is known to induce NEPHRIN and NEPH1 phosphorylation ( 3 , 8 , 30 ), we hypothesized that NEPHRIN and NEPH1 phosphorylation may require the downregulation of SHP-2 expression. Indeed, mRNA profiling of cultured podocytes injured by the puromycin amino nucleoside (Gene Expression Omnibus accession number GSE124622 ( 31 )) showed a 5-fold reduction in SHP-2 expression ( Fig. S5 ).…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of slit diaphragm proteins NEPHRIN and NEPH1 upon binding of HGF promotes podocyte repair

Solanki

Arif

Srivastava

et al. 2021

Journal of Biological Chemistry

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Phosphorylation (activation) and dephosphorylation (deactivation) of the slit diaphragm proteins NEPHRIN and NEPH1 are critical for maintaining the kidney epithelial podocyte actin cytoskeleton and, therefore, proper glomerular filtration. However, the mechanisms underlying these events remain largely unknown. Here we show that NEPHRIN and NEPH1 are novel receptor proteins for hepatocyte growth factor (HGF) and can be phosphorylated independently of the mesenchymal epithelial transition receptor in a ligand-dependent fashion through engagement of their extracellular domains by HGF. Furthermore, we demonstrate SH2 domain–containing protein tyrosine phosphatase-2–dependent dephosphorylation of these proteins. To establish HGF as a ligand, purified baculovirus-expressed NEPHRIN and NEPH1 recombinant proteins were used in surface plasma resonance binding experiments. We report high-affinity interactions of NEPHRIN and NEPH1 with HGF, although NEPHRIN binding was 20-fold higher than that of NEPH1. In addition, using molecular modeling we constructed peptides that were used to map specific HGF-binding regions in the extracellular domains of NEPHRIN and NEPH1. Finally, using an in vitro model of cultured podocytes and an ex vivo model of Drosophila nephrocytes, as well as chemically induced injury models, we demonstrated that HGF-induced phosphorylation of NEPHRIN and NEPH1 is centrally involved in podocyte repair. Taken together, this is the first study demonstrating a receptor-based function for NEPHRIN and NEPH1. This has important biological and clinical implications for the repair of injured podocytes and the maintenance of podocyte integrity.

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The Use of High-Throughput Transcriptomics to Identify Pathways with Therapeutic Significance in Podocytes

Cited by 10 publications

References 34 publications

The Role of miR-217-5p in the Puromycin Aminonucleoside-Induced Morphological Change of Podocytes

The Role of miR-217-5p in the Puromycin Aminonucleoside-Induced Morphological Change of Podocytes

Alternatively Spliced Landscape of PPARγ mRNA in Podocytes is Distinct from Adipose Tissue

Phosphorylation of slit diaphragm proteins NEPHRIN and NEPH1 upon binding of HGF promotes podocyte repair

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