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

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Publisher: Springer
Document Type: Report
Length: 499 words

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Byline: Tarak Srivastava (1), Mukut Sharma (2), Kok-Hooi Yew (3), Ram Sharma (2), R. Scott Duncan (3), Moin A. Saleem (4), Ellen T. McCarthy (5), Alexander Kats (6), Patricia A. Cudmore (1), Uri S. Alon (1), Christopher J. Harrison (3) Keywords: Innate immunity; Toll-like receptors; Cytokines; Lipopolysaccharide; Puromycin Aminonucleoside; Minimal Change Disease; Glomerular filtration barrier; Podocytes 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 ug/ml) or PAN (60 ug/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 4aY=2 3 6 5), the adapter molecule, MyD88, and transcription factor NF-IoB within one hour. LPS also caused increased levels of IL-6, IL-8 and MCP1 without exerting any effect on TNF-[alpha], IFN-[alpha] or TGF-[beta]1 at 24 h. Immunofluorescence intensity analysis of confocal microscopy images showed that LPS induced a significant increase in nuclear translocation of NF-IoB 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-IoB 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-IoB by PAN suggest that disparate but complementary mechanisms may contribute to the development of podocytopathy in MCD. Author Affiliation: (1) Section of Nephrology, Children's Mercy Hospital and University of Missouri at Kansas City, Kansas City, MO, USA (2) Renal Research Laboratory, Kansas City VA Medical Center, F2-120, Research Bldg 4801 E. Linwood Blvd, STOP 151, Kansas City, MO, 64128, USA (3) Section of Infectious Diseases, Children's Mercy Hospital and University of Missouri at Kansas City, Kansas City, MO, USA (4) Section of Nephrology, Bristol Royal Hospital for Children, Bristol, UK (5) Kidney Institute, University of Kansas Medical Center, Kansas City, KS, USA (6) Department of Pathology and Laboratory Medicine, Children's Mercy Hospital and University of Missouri at Kansas City, Kansas City, MO, USA Article History: Registration Date: 06/11/2012 Received Date: 18/05/2012 Accepted Date: 06/11/2012 Online Date: 17/11/2012

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Gale Document Number: GALE|A323559175