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

  • Tarak Srivastava
  • Mukut SharmaEmail author
  • Kok-Hooi Yew
  • Ram Sharma
  • R. Scott Duncan
  • Moin A. Saleem
  • Ellen T. McCarthy
  • Alexander Kats
  • Patricia A. Cudmore
  • Uri S. Alon
  • Christopher J. Harrison
Research Article

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.

Keywords

Innate immunity Toll-like receptors Cytokines Lipopolysaccharide Puromycin Aminonucleoside Minimal Change Disease Glomerular filtration barrier Podocytes 

Notes

Acknowledgements

The work was supported by The Paul Henson Endowed Fund for Pediatric Research and The Sam and Helen Kaplan Research Fund in Pediatric Nephrology. We thank Ashley Sherman, M.A. for assistance in statistical analysis. Parts of the work were presented at the XVth Congress of the International Pediatric Nephrology Association, New York, 30th August 2010.

Conflict of interest

None

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Copyright information

© US Government 2012

Authors and Affiliations

  • Tarak Srivastava
    • 1
  • Mukut Sharma
    • 2
    Email author
  • 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
  1. 1.Section of NephrologyChildren’s Mercy Hospital and University of Missouri at Kansas CityKansas CityUSA
  2. 2.Renal Research Laboratory, Kansas City VA Medical CenterKansas CityUSA
  3. 3.Section of Infectious DiseasesChildren’s Mercy Hospital and University of Missouri at Kansas CityKansas CityUSA
  4. 4.Section of NephrologyBristol Royal Hospital for ChildrenBristolUK
  5. 5.Kidney InstituteUniversity of Kansas Medical CenterKansas CityUSA
  6. 6.Department of Pathology and Laboratory MedicineChildren’s Mercy Hospital and University of Missouri at Kansas CityKansas CityUSA

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