LPS and PAN-induced podocyte injury in an in vitro model of minimal change disease: changes in TLR profile

Abstract

Minimal change disease (MCD), the most common idiopathic nephrotic syndrome in children, is characterized by proteinuria and loss of glomerular visceral epithelial cell (podocyte) ultrastructure. Lipopolysaccharide (LPS) and puromycin aminonucleoside (PAN) are used to study podocyte injury in models of MCD in vivo and in vitro. We hypothesized that LPS and PAN influence components of the innate immune system in podocytes such as the Toll-Like Receptor (TLRs), TLR adapter molecules, and associated cytokines. Our results show that cultured human podocytes constitutively express TLRs 1-6 and TLR-10, but not TLRs 7-9. LPS (25 μg/ml) or PAN (60 μg/ml) caused comparable derangement of the actin cytoskeleton in podocytes. Quantitative RT-PCR analysis show that LPS differentially up-regulated the expression of genes for TLRs (1 > 4 ≥ 2 > 3 > 6 > 5), the adapter molecule, MyD88, and transcription factor NF-κB within one hour. LPS also caused increased levels of IL-6, IL-8 and MCP1 without exerting any effect on TNF-α, IFN-α or TGF-β1 at 24 h. Immunofluorescence intensity analysis of confocal microscopy images showed that LPS induced a significant increase in nuclear translocation of NF-κB by 6 h. In contrast, PAN-induced only small changes in the expression of TLRs 2-6 that included a persistent increase in TLRs 2 and 5, a transient increase in TLR-4, and a gradual increase in TLRs 3 and 6 between 1 and 6 h. Correspondingly, it did not alter pro-inflammatory cytokine levels in podocytes. However, PAN induced a low but significant increase in NF-κB nuclear translocation within one hour that remained unchanged up to 6 h. In summary, these novel findings show that LPS, a known TLR-4 ligand, induced the gene expression of multiple TLRs with maximum effect on the expression of TLR-1 suggesting a loss of receptor selectivity and induction of receptor interactions in podocytes. A comparable derangement of the podocyte cytoskeleton and significant increase in the nuclear translocation of NF-κB by PAN suggest that disparate but complementary mechanisms may contribute to the development of podocytopathy in MCD.

Up-Regulation of CD74 Expression in Parietal Epithelial Cells in a Mouse Model of Focal Segmental Glomerulosclerosis

“…These findings indicate that the CD74-positive enlarged cells observed rapidly after LPS treatment reflected the amplification of PEC activation, which was already underway after ADR injection. Because LPS treatment is known to induce massive proteinuria via podocyte stimulation [21,32], we believe that PECs respond to podocyte injury with LPS treatment in ADR mice through morphological and phenotypic changes [33]. In addition to indirect effects through massive proteinuria with podocyte injury, a direct effect of LPS in PECs injury, which gradually progressed in ADR mice during the course of the treatment, should also be considered.…”

Slit Diaphragm Protein Neph1 and Its Signaling

“…The PAN-induced podocyte injury leads to actin cytoskeleton damage that is characterized by loss of fine F-actin stress fibers and the appearance of thick bundles of cortical actin filaments in podocytes (13,44). In vivo PAN treatment has been shown to induce foot process effacement along with the decreased expression and abnormal distribution of the slit diaphragm proteins Neph1, nephrin, and podocin (14,15,50).…”

“…5A). The cytoskeletal damage is often characterized by a gradual increase in stress fibers and retraction of cellular processes with PAN treatment (13,15,44). Eventually in a time-dependent manner, the stress actin fibers started moving toward the cell periphery, and the cell-cell contacts were lost (Figs.…”

Urinary Xist is a potential biomarker for membranous nephropathy

“…The E11 cell lines, previously described in detail [17], were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), and penicillin/streptomycin at 33°C under a humidified atmosphere of 5% CO 2 . E11 cells at 50% confluence were thermoswitched from 33°C to 38°C for 14 days.…”