C-Peptide Signals via G i to Protect against TNF-– Mediated Apoptosis of Opossum Kidney Proximal Tubular Cells Nawal M. Al-Rasheed,* Gary B. Willars,* and Nigel J. Brunskill †‡ *Department of Cell Physiology and Pharmacology, and † Department of Infection, Immunity and Inflammation, University of Leicester School of Medicine; ‡ Department of Nephrology, Leicester General Hospital, Leicester, United Kingdom Cell loss by apoptosis occurs in renal injury such as diabetic nephropathy. TNF- 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-–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-–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-. Compromised cell viability was reversed by pretreatment with 5 nM C-peptide or 100 nM insulin. TNF-–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- 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-B. Activation of NF-B by C-peptide was pertussis toxin sensitive and dependent on activation of G i . Phosphatidylinositol 3-kinase but not extracellular signal regulated mitogen-activated protein kinase mediated C-peptide and insulin activation of NF-B. 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-B– dependent survival gene. These data suggest that C-peptide and/or insulin activation of NF-B–regulated survival genes protects against TNF-–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. J Am Soc Nephrol 17: 986 –995, 2006. doi: 10.1681/ASN.2005080797 C -peptide, a cleavage product that is derived from the proinsulin molecule in the course of insulin biosyn- thesis (1), is stored in the secretory granules of pan- creatic  cells and is released eventually into the portal circu- lation in an amount equimolar with that of insulin (1). Although C-peptide has an important clinical role as a surro- gate marker of insulin release, it generally is regarded as bio- logically inert. Until recently, popular dogma held that the only significant physiologic function of C-peptide related to facilita- tion of proinsulin folding to allow the accurate alignment of the A and B chains of insulin (1). However, a paradigm shift in thinking is occurring. C-peptide is undergoing a reevaluation as a peptide hormone in its own right, independent of insulin, possibly acting through a G protein– coupled membrane recep- tor (2). Evolving evidence suggests that C-peptide might have a protective role in diabetes by ameliorating diabetic complica- tions (3– 6). C-peptide has been shown to exert beneficial effects on both renal function and morphology in diabetic nephropa- thy (4,7,8). These observations are supported by the findings that pancreas transplantation may induce reversal of diabetic nephropathy (9) and that patients who have type 1 diabetes and receive combined kidney/islet cell transplants have a better renal prognosis than patients who receive a kidney transplant alone (10), irrespective of metabolic control. The mechanisms that underlie these beneficial effects of C- peptide are incompletely understood. However, C-peptide has been found to stimulate numerous intracellular signaling path- ways in proximal tubular cells such as mitogen-activated pro- tein kinases, phosphatidylinositol 3-kinase (PI3-K)/Akt, and protein kinase C (11), resulting in increases in intracellular [Ca 2+ ] (11,12). C-peptide also has been shown to stimulate endothelial nitric oxide synthase in endothelial cells (13) and Na + ,K + -ATPase activity in both glomerular and tubular cells (14). TNF- is a pleiotropic 157–amino acid peptide cytokine that is capable of eliciting a wide spectrum of cellular responses, including differentiation, proliferation, inflammation, and cell death (15), via interaction with two members of the TNF recep- tor family, TNF-R1 and TNF-R2. Predominantly produced by monocytes/macrophages but also by T and B lymphocytes and Received August 1, 2005. Accepted January 10, 2006. Published online ahead of print. Publication date available at www.jasn.org. Address correspondence to: Dr. Nigel J Brunskill, Department of Nephrology, Leicester General Hospital, Gwendolen Road, Leicester LE5 4PW, UK. Phone: +44-116-258-8043; Fax: +44-116-258-4764; E-mail: njb18@le.ac.uk Copyright © 2006 by the American Society of Nephrology ISSN: 1046-6673/1704-0986