ABSTRACT
Glomerular diseases are the leading cause of chronic kidney diseases with the pathomechanisms largely unclear. ANGPT2 is known to regulate endothelial cell homeostasis through TEK/Tie2 and its dysregulation causes endothelial damage. Here, we found that ANGPT2 is upregulated in glomerular diseases and wondered whether it also acts on the other two glomerular cell types, podocytes and mesangial cells. We treated podocytes and mesangial cells in culture with ANGPT2 but didn't find changes in morphology and survival. RNA-seq analysis revealed that gene expression was altered in both podocytes and mesangial cells and that the differentially expressed genes in the two cell types were fundamentally different and enriched in distinct cellular processes and pathways according to GO and KEGG analyses. Mechanistically, the Ingenuity Pathway Analysis (IPA) analysis revealed that ERK and AKT were the most connected nodes in the networks of the regulated genes of both podocytes and mesangial cells, suggesting that ANGPT2 affected ERK and AKT in both cell types. Interestingly, immunoblotting showed that phosphorylated ERK and AKT were both increased in podocytes while decreased in mesangial cells by ANGPT2. We found that mesangial cells, but not podocytes, expressed TEK and ANGPT1, suggesting that ANGPT2 could antagonize ANGPT1-TEK-ERK axis in mesangial cells similarly to endothelial cells. We searched databases and found that integrin alpha(v) (ITGAV) is an ANGPT2 interacting protein and expressed in podocytes, suggesting that ITGAV mediates ANGPT2 effect on podocytes. In conclusion, increased ANGPT2 may be involved in glomerular injury by affecting podocytes and mesangial cells in addition to endothelial cells. The complexity of the effect of ANGPT2 in glomeruli may apply to other factors.
ABSTRACT
MicroRNAs (miRNAs) are emerging as effective therapeutic agents. When testing whether miR-145-5p could alleviate kidney injury, we unexpectedly found that extracellular vesicles loaded with miR-145-5p induced proteinuria and podocyte foot process effacement in normal control mice. To explore the mechanism of miR-145-5p's toxicity to podocytes, we hypothesized that miR-145-5p could enter podocytes and inhibit genes essential for podocytes. We demonstrated that systemically administered miRNA can enter podocytes. Next, we predicted 611 podocyte essential genes based on single-cell RNA sequencing (RNA-seq) and found that 32 of them are predicted to be targeted by miR-145-5p. Functional annotation of the 32 podocyte essential genes revealed small GTPase-mediated signal transduction as the top pathway. We experimentally validated that miR-145-5p targeted Arhgap24 and Srgap1, the essential regulators of the Rho family of small GTPases, increased the activity of Rac1 and Cdc42, and reduced RhoA activity, accompanied by cellular injury, in podocytes. These results explain how miR-145-5p has deleterious effect on podocytes. Most importantly, our study provides a novel approach to investigate how a miRNA affects a given cell type, allowing not only identification of the molecular mechanism underlying an observed side effect of a miRNA drug but also prediction of miRNA drug toxicity on various cell types.
ABSTRACT
Pofut1 gene encodes a O-fucosyltransferase that adds fucose to the serine/threonine residue in the sequence of C2XXXX(S/T)C3 of EGF-like domain in a protein. O-fucosylation has been shown to be required for some EGF-like domain-containing proteins to function, e.g., Notch1, and POFUT1 deficiency could affect cellular function and cause diseases. Pofut1 is ubiquitously expressed, but its essentiality for most cell types is not known. In the present study, we examined the consequence of Pofut1 gene abrogation in mouse podocytes using Cre-loxP system, and found that the conditional knockout mice were indistinguishable from wild-type controls in urinary protein level, glomerular morphology, podocyte foot process ultrastructure, podocyte marker expression and podocyte numbers. These results indicated that POFUT1 is not essential for podocyte structure, function and survival in mice. To understand why POFUT1 is dispensable for podocytes, we searched mouse podocyte essential gene candidates (as determined by single-cell RNA-seq) and found only two POFUT1 substrates, NOTCH2 and tPA. It has been shown that abrogation of these genes does not cause podocyte injury, explaining dispensability of POFUT1 for mouse podocytes and demonstrating a feasibility to predict POFUT1 essentiality for a given cell type. At present, most mouse cell types have been subject to single-cell RNA-seq, making essential gene prediction and thus POFUT1 requirement prediction possible for the cell types.
