The expression of podocyte-specific proteins in parietal epithelial cells is regulated by protein degradation

Abstract

The role of parietal epithelial cells (PECs) in glomerular disease is unclear because they also express podocyte proteins under pathophysiological conditions. To help resolve this, we established a novel PEC isolation technique in rats and mice to investigate which regulatory mechanisms lead to podocyte protein expression in PECs. This pure pool of naive PECs was then compared with PECs in primary culture and immortalized PECs in permanent culture. The naive PECs expressed low levels of podocyte-specific mRNA. Accordingly, in crescentic glomerulonephritis, single PECs activated the podocin promoter in vivo. In primary culture, PECs expressed a distinct morphology from podocytes but with high transcript and protein levels of PEC markers. In contrast to naive PECs, cultured PECs also expressed podocyte proteins, and this correlated with reduced proteolytic activity but not with increased transcript levels. Activation of autophagy or proteasomal degradation decreased the levels of podocyte proteins in PECs, whereas inhibition of proteasomal degradation led to the stabilization of podocyte proteins in PECs. Thus, naive PECs express podocyte transcripts physiologically and these podocyte proteins are stable under pathological conditions through decreased proteolysis.

MicroRNA-193a Regulates the Transdifferentiation of Human Parietal Epithelial Cells toward a Podocyte Phenotype

“…To circumvent these limitations, we confirmed our results obtained in hPECs with experiments in immortalized rat PECs. 13 On inhibition of miR-193a with complementary locked nucleic acids (LNAs), 15 rat PECs arborized and expressed high levels of podocyte proteins compared with control rat PECs treated with scrambled LNA (Supplemental Figure 2). In summary, we could successfully show a conversion of PECs to podocytes in vitro on KD or inhibition of miR-193a.…”

“…We could, furthermore, show that expression of podocyte proteins was negatively regulated through UCH-L1/the ubiquitin proteasome and the autophagosomal/lysosomal degradation system in an immortalized rat PEC cell line 13 and cultured human podocytes. 14 However, inhibition of neither UCH-L1 nor the proteasome was sufficient to induce a full PEC-to-podocyte conversion 13 (C. MeyerSchwesinger, unpublished observation). This suggests the existence of an additional layer of regulation, which could be performed by a microRNA (miR).…”

“…The hPEC line was established with the help of a manual dissection technique of BCs that had previously led to the successful generation of a rat polyclonal PEC line. 13 The hPECs resembled cultured mouse and rat PECs 13 with respect to cellular morphology, growth characteristics, and gene expression profiles.…”

“…16 As described in detail in Concise Methods, BCs were gently dissected off the glomerular tufts with minutia pins under visual control. 13 hPECs were allowed to adhere to and proliferate on collagen-1-coated dishes (Scheme 1). The adhering primary hPECs exhibited a clonal regular cobblestone morphology ( Figure 1A, A).…”

“…[8][9][10][11][12] In our recent work, we found low levels of podocyte-specific transcripts in naïve PECs of rats, 13 suggesting that PECs had the transcriptional prerequisite to express podocyte markers. We could, furthermore, show that expression of podocyte proteins was negatively regulated through UCH-L1/the ubiquitin proteasome and the autophagosomal/lysosomal degradation system in an immortalized rat PEC cell line 13 and cultured human podocytes. 14 However, inhibition of neither UCH-L1 nor the proteasome was sufficient to induce a full PEC-to-podocyte conversion 13 (C. MeyerSchwesinger, unpublished observation).…”