Combined expression of A1 and A20 achieves optimal protection of renal proximal tubular epithelial cells.
BACKGROUND
Apoptotic death of renal proximal tubular epithelial cells (RPTECs) is a feature of acute and chronic renal failure. RPTECs are directly damaged by ischemia, inflammatory, and cytotoxic mediators but also contribute to their own demise by up-regulating proinflammatory nuclear factor-kappaB (NF-kappaB)-dependent proteins. In endothelial cells, the Bcl family member A1 and the zinc finger protein A20 have redundant and dual antiapoptotic and anti-inflammatory effects. We studied the function(s) of A1 and A20 in human RPTECs in vitro.


METHODS
Expression of A1 [reverse transcription-polymerase chain reaction (RT-PCR) and A20 (Northern and Western blot analysis)] in RPTECs was evaluated. A1 and A20 were overexpressed in RPTECs by recombinant adenoviral-mediated gene transfer. Their effect upon inhibitor of NFkappaB alpha (IkappaBalpha) degradation (Western blot), NF-kappaB nuclear translocation [electrophoretic mobility shift assay (EMSA)], up-regulation of intercellular adhesion molecule-1 (ICAM-1) [fluorescence-activated cell sorter (FACS)] and monocyte chemoattractant protein-1 (MCP-1) (Northern blot) and apoptosis [terminal deoxynucleotiddyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL)] and FACS analysis of DNA content) was determined.


RESULTS
A1 and A20 were induced in RPTECs as part of the physiologic response to tumor necrosis factor (TNF). A20, but not A1, inhibited TNF-induced NF-kappaB activation by preventing IkappaBalpha degradation, hence subsequent up-regulation of the proinflammatory molecules ICAM-1 and MCP-1. Unexpectedly, A20 did not protect RPTECs from TNF and Fas-mediated apoptosis while A1 protected against both stimuli. Coexpression of A1 and A20 in RPTECs achieved additive anti-inflammatory and antiapoptotic cytoprotection.


CONCLUSIONS
A1 and A20 exert differential cytoprotective effects in RPTECs. A1 is antiapoptotic. A20 is anti-inflammatory via blockade of NF-kappaB. We propose that A1 and A20 are both required for optimal protection of RPTECs from apoptosis (A1) and inflammation (A20) in conditions leading to renal damage.
Kunter
Uta
U
Division of Vascular Surgery, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Daniel
Soizic
S
Arvelo
Maria B
MB
Choi
Jean
J
Shukri
Tala
T
Patel
Virendra I
VI
Longo
Christopher R
CR
Scali
Salvatore T
ST
Shrikhande
Gautam
G
Rocha
Eduardo
E
Czismadia
Eva
E
Mottley
Christina
C
Grey
Shane T
ST
Floege
Jürgen
J
Ferran
Christiane
C
D017209:Apoptosis / Q000502:physiology*; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000235:genetics; D004268:DNA-Binding Proteins; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology*; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics; D047908:Intracellular Signaling Peptides and Proteins; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000502:physiology*; D015778:Minor Histocompatibility Antigens; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D009393:Nephritis / Q000473:pathology / Q000503:physiopathology; D009687:Nuclear Proteins; D011506:Proteins / Q000235:genetics*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D015854:Up-Regulation; D019014:fas Receptor / Q000378:metabolism
C096816:BCL2-related protein A1; C111835:CCL2 protein, human; D018932:Chemokine CCL2; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D047908:Intracellular Signaling Peptides and Proteins; D015778:Minor Histocompatibility Antigens; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D009687:Nuclear Proteins; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor; D018799:Intercellular Adhesion Molecule-1; D000072000:NF-KappaB Inhibitor alpha; C076529:TNFAIP3 protein, human; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3