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

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Journal of Cell Communication and Signaling Aims and scope

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.

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References

  • Adeline M, Hajjar D, O’Mahony S, Ozinsky A, Underhill DM, Aderem A, Klebanoff SJ, Wilson CB (2001) Functional interactions between Toll-Like Receptor (TLR) 2 and TLR1 or TLR6 in response to phenol-soluble modulin. J Immunol 166:15–19

    Google Scholar 

  • Anders HJ, Schlöndorff D (2007) Toll-like receptors: emerging concepts in kidney disease. Curr Opin Nephrol Hypertens 16:177–83

    Article  PubMed  CAS  Google Scholar 

  • Araya CE, Wasserfall CH, Brusko TM, Mu W, Segal MS, Johnson RJ et al (2006) A case of unfulfilled expectations. Cytokines in idiopathic minimal lesion nephrotic syndrome. Pediatr Nephrol 21:603–610

    Article  PubMed  Google Scholar 

  • Banas MC, Banas B, Hudkins KL, Wietecha TA, Iyoda M, Bock E et al (2008) TLR4 links podocytes with the innate immune system to mediate glomerular injury. J Am Soc Nephrol 19:704–713

    Article  PubMed  CAS  Google Scholar 

  • Brown HJ, Lock HR, Wolfs TG, Buurman WA, Sacks SH, Robson MG (2007) Toll-like receptor 4 ligation on intrinsic renal cells contributes to the induction of antibody-mediated glomerulonephritis via CXCL1 and CXCL2. J Am Soc Nephrol 18:1732–1739

    Article  PubMed  CAS  Google Scholar 

  • Carmody RJ, Chen YH (2007) Nuclear factor-kappaB activation and regulation during toll-like receptor signaling. Cell Mol Immunol 4:31–41

    PubMed  CAS  Google Scholar 

  • Coers W, Huitema S, van der Horst ML, Weening JJ (1994) Puromycin aminonucleoside and adriamycin disturb cytoskeletal and extracellular matrix protein organization, but not protein synthesis of cultured glomerular epithelial cells. Exp Nephrol 2:40–50

    PubMed  CAS  Google Scholar 

  • Couser WG (2012) Basic and translational concepts of immune-mediated glomerular disease. J Am Soc Nephrol 23:381–399

    Article  PubMed  CAS  Google Scholar 

  • Diamond JR, Bonventre JV, Karnovsky MJ (1986) A role for oxygen free radicals in aminonucleoside nephrosis. Kidney Int 29:478–483

    Article  PubMed  CAS  Google Scholar 

  • Ding X, Zhu F, Li T, Zhou Q, Hou FF, Nie J (2011) Numb protects renal proximal tubular cells from puromycin aminonucleoside-induced apoptosis through inhibiting Notch signaling pathway. Int J Biol Sci 7:269–278

    Article  PubMed  CAS  Google Scholar 

  • Farhat K, Riekenberg S, Heine H, Debarry J, Lang R, Mages J et al (2008) Heterodimerization of TLR2 with TLR1 or TLR6 expands the ligand spectrum but does not lead to differential signaling. J Leukoc Biol 83:692–701

    Article  PubMed  CAS  Google Scholar 

  • Frenk S, Antonowicz I, Craig JM, Metcoff J (1955) Experimental nephrotic syndrome induced in rats by aminonucleoside; renal lesions and body electrolyte composition. Proc Soc Exp Biol Med 89:424–427

    PubMed  CAS  Google Scholar 

  • Gloy J, Reitinger S, Fischer K–G, Schreiber R, Boucherot A, Kunzelmann K et al (2000) Amino acid transport in mouse podocytes. Am J Physiol 278:F999–F1005

    CAS  Google Scholar 

  • Greiber S, Müller B, Daemisch P, Pavenstädt H (2002) Reactive oxygen species alter gene expression in podocytes: induction of granulocyte macrophage-colony-stimulating factor. J Am Soc Nephrol 13:86–95

    PubMed  CAS  Google Scholar 

  • Gribar SC, Richardson WM, Sodhi CP, Hackam DJ (2008) No longer an innocent bystander: epithelial toll-like receptor signaling in the development of mucosal inflammation. Mol Med 14:645–659

    Article  PubMed  CAS  Google Scholar 

  • Ishimoto T, Shimada M, Araya CE, Huskey J, Garin EH, Johnson RJ (2011) Minimal Change Disease: A CD80 podocytopathy? Semin Nephrol 31:320–325

    Article  PubMed  CAS  Google Scholar 

  • Kistler AD, Reiser J (2010) Maximal ‘CD80-uria’ with minimal change. Kidney Int 78:236–238

    Article  PubMed  CAS  Google Scholar 

  • Lane JC, Kaskel FJ (2009) Pediatric nephrotic syndrome: from the simple to the complex. Semin Nephrol 29:389–398

    Article  PubMed  CAS  Google Scholar 

  • Lannigan R (1963) The production of chronic renal disease in rats by a single intravenous injection of aminonucleoside of puromycin and the effect of low dosage continuous hydrocortisone. Br J Exp Pathol 44:326–333

    PubMed  CAS  Google Scholar 

  • MacDonald NE, Wolfish N, McLaine P, Phipps P, Rossier E (1986) Role of respiratory viruses in exacerbations of primary nephrotic syndrome. J Pediatr 108:378–382

    Article  PubMed  CAS  Google Scholar 

  • Marshall CB, Pippin JW, Krofft RD, Shankland SJ (2006) Puromycin aminonucleoside induces oxidant-dependent DNA damage in podocytes in vitro and in vivo. Kidney Int 70:1962–1973

    Article  PubMed  CAS  Google Scholar 

  • Mathieson PW (2007) Minimal change nephropathy and focal segmental glomerulosclerosis. Semin Immunopathol 29:415–426

    Article  PubMed  Google Scholar 

  • Matsumoto M, Funami K, Tanabe M, Oshiumi H, Shingai M, Seto Y et al (2003) Subcellular localization of Toll-like receptor 3 in human dendritic cells. J Immunol 171:3154–62

    PubMed  CAS  Google Scholar 

  • McCarthy ET, Sharma R, Sharma M, Li JZ, Ge X, Dileepan KN et al (1998) Tumor Necrosis Factor-α increases albumin permeability of isolated rat glomeruli through the generation of superoxide. J Amer Soc Nephrol 9:433–438

    CAS  Google Scholar 

  • Medzhitov R, Preston-Hurlburt P, Janeway CA Jr (1997) A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388:394–397

    Article  PubMed  CAS  Google Scholar 

  • Neuhaus TJ, Fay J, Dillon MJ, Trompeter RS, Barratt TM (1994) Alternative treatment to corticosteroids in steroid sensitive idiopathic nephrotic syndrome. Arch Dis Child 71:522–526

    Article  PubMed  CAS  Google Scholar 

  • Nishiya T, DeFranco AL (2004) Ligand-regulated chimeric receptor approach reveals distinctive subcellular localization and signaling proper ties of the Toll-like receptors. J Biol Chem 279:19008–19017

    Article  PubMed  CAS  Google Scholar 

  • Ospelt C, Gay S (2010) TLRs and chronic inflammation. Int J biochem Cell Biol 42:495–505

    Article  PubMed  CAS  Google Scholar 

  • Pawar RD, Castrezana-Lopez L, Allam R, Kulkarni OP, Segerer S, Radomska E et al (2009) Bacterial lipopeptide triggers massive albuminuria in murine lupus nephritis by activating Toll-like receptor 2 at the glomerular filtration barrier. Immunology 128(1 Suppl):e206–221

    Article  PubMed  Google Scholar 

  • Pippin JW, Brinkkoetter PT, Cormack-Aboud FC, Durvasula RV, Hauser PV, Kowalewska J et al (2009) Inducible rodent models of acquired podocyte diseases. Am J Physiol Renal Physiol 296:F213–229

    Article  PubMed  CAS  Google Scholar 

  • Reiser J, von Gersdorff G, Loos M, Oh J, Asanuma K, Giardino L et al (2004) Induction of B7-1 in podocytes is associated with nephrotic syndrome. J Clin Invest 113:1390–1397

    PubMed  CAS  Google Scholar 

  • Saha TC, Singh H (2006) Minimal Change Disease: A Review. Southern Med J 99:1264–1270

    Article  PubMed  Google Scholar 

  • Saleem MA, O’Hare MJ, Reiser J, Coward RJ, Inward CD, Farren T et al (2002) A conditionally immortalized human podocyte cell line demonstrating nephrin and podocin expression. J Am Soc Nephrol 13:630–638

    PubMed  CAS  Google Scholar 

  • Sanchez-Niño MD, Benito-Martin A, Gonçalves S, Sanz AB, Ucero AC, Izquierdo MC et al (2010) TNF superfamily: a growing saga of kidney injury modulators. Mediators Inflamm. doi:10.1055/2010/182958

  • Savin VJ, Sharma M, McCarthy ET, Sharma R, Reddy S, Dong JW et al (2008) Cardiotrophin like cytokine-1: Candidate for the focal glomerular sclerosis permeability factor. J Am Soc Neph 19:59A

    Article  Google Scholar 

  • Sellier-Leclerc AL, Duval A, Riveron S, Macher MA, Deschenes G, Loirat C et al (2007) A humanized mouse model of idiopathic nephrotic syndrome suggests a pathogenic role for immature cells. J Am Soc Nephrol 18:2732–2739

    Article  PubMed  Google Scholar 

  • Shiiki H, Sasaki Y, Nishino T, Kimura T, Kurioka H, Fujimoto S et al (1998) Cell proliferation and apoptosis of the glomerular epithelial cells in rats with puromycin aminonucleoside nephrosis. Pathobiology 66:221–229

    Article  PubMed  CAS  Google Scholar 

  • Shimada M, Araya C, Rivard C, Ishimoto T, Johnson RJ, Garin EH (2011) Minimal change disease: a “two-hit” podocyte immune disorder? Pediatr Nephrol 26:645–649

    Article  PubMed  Google Scholar 

  • Shimada M, Ishimoto T, Lee PY, Lanaspa MA, Rivard CJ, Roncal-Jimenez CA et al (2012) Toll-like receptor 3 ligands induce CD80 expression in human podocytes via an NF-{kappa}B-dependent pathway. Nephrol Dial Transplant 27:81–89

    Article  PubMed  CAS  Google Scholar 

  • Smoyer WE, Ransom RF (2002) Hsp27 regulates podocyte cytoskeletal changes in an in vitro model of podocyte process retraction. FASEB J 16:315–326

    Article  PubMed  CAS  Google Scholar 

  • Spitzer JH, Visintin A, Mazzoni A, Kennedy MN, Segal DM (2002) Toll-like receptor 1 inhibits Toll-like receptor 4 signaling in endothelial cells. Eur J Immunol 32:1182–1187

    Article  PubMed  CAS  Google Scholar 

  • Srivastava T, Simon SD, Alon US (1999) High incidence of focal segmental glomerulosclerosis in nephrotic syndrome of childhood. Pediatr Nephrol 13:13–18

    Article  PubMed  CAS  Google Scholar 

  • Srivastava T, McCarthy ET, Sharma R, Cudmore PA, Sharma M, Johnson ML et al (2010) Prostaglandin E2 is crucial in the response of podocytes to fluid flow shear stress. J Cell Commun Signal 4:79–90

    Article  PubMed  Google Scholar 

  • Sun Y, He L, Takemoto M, Patrakka J, Pikkarainen T, Genové G, Norlin J et al (2009) Glomerular transcriptome changes associated with lipopolysaccharide-induced proteinuria. Am J Nephrol 29:558–570

    Article  PubMed  CAS  Google Scholar 

  • Suranyi MG, Guasch A, Hall BM, Myers BD (1993) Elevated levels of tumor necrosis factor-alpha in the nephrotic syndrome in humans. Am J Kidney Dis 21:251–259

    PubMed  CAS  Google Scholar 

  • Suzuki T, Takemura H, Noiri E, Nosaka K, Toda A, Taniguchi S et al (2001) Puromycin aminonucleoside induces apoptosis and increases HNE in cultured glomerular epithelial cells. Free Radic Biol Med 31:615–623

    Article  PubMed  CAS  Google Scholar 

  • Takeda K, Akira S (2004) TLR signaling pathways. Sem Immunol 16:3–9

    Article  CAS  Google Scholar 

  • Tipping PG (2008) Are podocytes passive or provocative in proteinuric glomerular pathology? J Am Soc Nephrol 19:651–653

    Article  PubMed  Google Scholar 

  • Van Den Berg JG, Aten J, Chand MA, Claessen N, Dijkink L et al (2000) Interleukin-4 and interleukin-13 act on glomerular visceral epithelial cells. J Am Soc Nephrol 11:413–422

    Google Scholar 

  • Walia D, Sharma M, Raveendran V, Zhou J, Sharma R, Stechschulte DJ et al (2012) Human Mast Cells (HMC-1 C56) Enhance Interleukin-6 production by quiescent and lipopolysaccharide-stimulated human coronary artery endothelial cells. Mediators Inflamm. doi:10.1155/2012/274347

  • Wasilewska A, Zoch-Zwierz W, Taranta-Janusz K, Kołodziejczyk Z (2011) Urinary monocyte chemoattractant protein-1 excretion in children with glomerular proteinuria. Scand J Urol Nephrol 45:52–59

    Article  PubMed  CAS  Google Scholar 

  • Woroniecki RP, Shatat IF, Supe K, Du Z, Kaskel FJ (2008) Urinary cytokines and steroid responsiveness in idiopathic nephrotic syndrome of childhood. Am J Nephrol 28:83–90

    Article  PubMed  CAS  Google Scholar 

  • Yamamoto M, Sato S, Hemmi H, Uematsu S, Hoshino K, Kaisho T et al (2003) TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent signaling pathway. Nat Immunol 4:1144–1150

    Article  PubMed  CAS  Google Scholar 

  • Yap HK, Cheung W, Murugasu B, Sim SK, Seah CC, Jordan SC (1999) Th1 and Th2 cytokine mRNA profiles in childhood nephrotic syndrome: evidence for increased IL-13 mRNA expression in relapse. J Am Soc Nephrol 10:529–537

    PubMed  CAS  Google Scholar 

  • Yew KH, Carsten B, Harrison C (2010) Scavenger receptor A1 is required for sensing HCMV by endosomal TLR-3/-9 in monocytic THP-1 cells. Mol Immunol 47:883–893

    Article  PubMed  CAS  Google Scholar 

  • Zarember KA, Godowski PJ (2002) Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J Immunol 168:554–561

    PubMed  CAS  Google Scholar 

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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.

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Authors and Affiliations

  1. Section of Nephrology, Children’s Mercy Hospital and University of Missouri at Kansas City, Kansas City, MO, USA

    Tarak Srivastava, Patricia A. Cudmore & Uri S. Alon

  2. Renal Research Laboratory, Kansas City VA Medical Center, F2-120, Research Bldg 4801 E. Linwood Blvd, STOP 151, Kansas City, MO, 64128, USA

    Mukut Sharma & Ram Sharma

  3. Section of Infectious Diseases, Children’s Mercy Hospital and University of Missouri at Kansas City, Kansas City, MO, USA

    Kok-Hooi Yew, R. Scott Duncan & Christopher J. Harrison

  4. Section of Nephrology, Bristol Royal Hospital for Children, Bristol, UK

    Moin A. Saleem

  5. Kidney Institute, University of Kansas Medical Center, Kansas City, KS, USA

    Ellen T. McCarthy

  6. Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital and University of Missouri at Kansas City, Kansas City, MO, USA

    Alexander Kats

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  1. Tarak Srivastava
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Correspondence to Mukut Sharma.

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Srivastava, T., Sharma, M., Yew, KH. et al. LPS and PAN-induced podocyte injury in an in vitro model of minimal change disease: changes in TLR profile. J. Cell Commun. Signal. 7, 49–60 (2013). https://doi.org/10.1007/s12079-012-0184-0

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