title: C-peptide signals via Galpha i to protect against TNF-alpha-mediated apoptosis of opossum kidney proximal tubular cells.
abstract: Cell loss by apoptosis occurs in renal injury such as diabetic nephropathy. TNF-alpha is a cytokine that induces apoptosis and has been implicated in the pathogenesis of diabetic nephropathy. The aim was to investigate whether C-peptide or insulin could modulate TNF-alpha-mediated cell death in opossum kidney proximal tubular cells and to examine the mechanism(s) of any effects observed. C-peptide and insulin protect against TNF-alpha-induced proximal tubular cell toxicity and apoptosis. Cell viability was analyzed by methylthiazoletetrazolium assay; cell viability was reduced to 60.8 +/- 2.7% of control after stimulation with 300 ng/ml TNF-alpha. Compromised cell viability was reversed by pretreatment with 5 nM C-peptide or 100 nM insulin. TNF-alpha-induced apoptosis was detected by DNA nick-end labeling and by measuring histone associated DNA fragments using ELISA. By ELISA assay, 300 ng/ml TNF-alpha increased apoptosis by 145.8 +/- 4.9% compared with controls, whereas 5 nM C-peptide and 100 nM insulin reduced apoptosis to 81.6 +/- 4.8 and 77.4 +/- 3.1% of control, respectively. The protective effects of C-peptide and insulin were associated with activation of NF-kappaB. Activation of NF-kappaB by C-peptide was pertussis toxin sensitive and dependent on activation of Galpha(i). Phosphatidylinositol 3-kinase but not extracellular signal regulated mitogen-activated protein kinase mediated C-peptide and insulin activation of NF-kappaB. The cytoprotective effects of both C-peptide and insulin were related to increased expression of TNF receptor-associated factor 2, the product of an NF-kappaB-dependent survival gene. These data suggest that C-peptide and/or insulin activation of NF-kappaB-regulated survival genes protects against TNF-alpha-induced renal tubular injury in diabetes. The data further support the concept of C-peptide as a peptide hormone in its own right and suggest a potential therapeutic role for C-peptide.
authors.affiliation: Department of Nephrology, Leicester General Hospital, Gwendolen Road, Leicester LE5 4PW, UK.
authors.forename: Nawal M
authors.initials: NM
authors.lastname: Al-Rasheed
authors.forename: Gary B
authors.initials: GB
authors.lastname: Willars
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Brunskill
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002096:C-Peptide / Q000378:metabolism* / Q000494:pharmacology; D002460:Cell Line; D003928:Diabetic Nephropathies / Q000209:etiology / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D009893:Opossums; D015398:Signal Transduction; D047992:TNF Receptor-Associated Factor 2 / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D002096:C-Peptide; D007328:Insulin; D007976:Leupeptins; D016328:NF-kappa B; D047992:TNF Receptor-Associated Factor 2; D014409:Tumor Necrosis Factor-alpha; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde