Modulation of Growth Hormone Signal Transduction in Kidneys of Streptozotocin-Induced Diabetic Animals Effect of a Growth Hormone Receptor Antagonist Ana C.P. Thirone, John A. Scarlett, Alessandra L. Gasparetti, Eliana P. Araujo, Maria H.L. Lima, Carla R.O. Carvalho, Lı ´cio A. Velloso, and Mario J.A. Saad Growth hormone (GH) and IGFs have a long distin- guished history in diabetes, with possible participation in the development of renal complications. The impli- cated effect of GH in diabetic end-stage organ damage may be mediated by growth hormone receptor (GHR) or postreceptor events in GH signal transduction. The present study investigates the effects of diabetes in- duced by streptozotocin (STZ) on renal GH signaling. Our results demonstrate that JAK2, insulin receptor substrate (IRS)-1, Shc, ERKs, and Akt are widely dis- tributed in the kidney, and after GH treatment, there is a significant increase in phosphorylation of these pro- teins in STZ-induced diabetic rats compared with con- trols. Moreover, the GH-induced association of IRS-1/ phosphatidylinositol 3-kinase, IRS-1/growth factor receptor bound 2 (Grb2), and Shc/Grb2 are increased in diabetic rats as well. Immunohistochemical studies show that GH-induced p-Akt and p-ERK activation is apparently more pronounced in the kidneys of diabetic rats. Administration of G120K-PEG, a GH antagonist, in diabetic mice shows inhibitory effects on diabetic renal enlargement and reverses the alterations in GH signal transduction observed in diabetic animals. The present study demonstrates a role for GH signaling in the pathogenesis of early diabetic renal changes and sug- gests that specific GHR blockade may present a new concept in the treatment of diabetic kidney disease. Diabetes 51:2270 –2281, 2002 D iabetic nephropathy is one of the most common causes of end-stage renal failure in the Western world (1,2). Renal and glomerular hypertro- phies are observed in the early phase of human and experimental diabetes (3,4), and the search for signif- icant pathogenic mechanisms in diabetic kidney disease has focused on these early events. Growth hormone (GH) and IGFs have a long and distinguished history in diabetes, with possible participation in the development of renal complications (5,6). Accordingly, in early experimental diabetes, an initial transient increase in kidney IGF-1 is observed, followed by renal and glomerular hypertrophy (7,8). Also, diabetic dwarf rats are characterized by dimin- ished renal and glomerular hypertrophy compared with diabetic control animals with intact pituitary (9,10), and administration of the long-acting somatostatin analog oc- treotide to streptozotocin (STZ)-induced diabetic animals inhibits renal and glomerular hypertrophy (7). In addition, specific changes occur in the renal GH binding protein mRNA, IGF-1 receptor mRNA, and IGF binding protein mRNA expression in long-term diabetes (5–10). The growth hormone receptor (GHR) itself is not a tyrosine kinase (11), but after receptor binding, JAK2, a member of the Janus family of tyrosine kinases, is acti- vated after its association with a dimerized GHR in re- sponse to hormone binding (12). As a consequence of the kinase activation, GH stimulates the tyrosyl phosphoryla- tion of some substrates, such as insulin receptor substrate (IRS)-1 (13–16), IRS-2 (16,17), and Shc proteins (16,18). Tyrosyl phosphorylation of IRSs in response to GH pro- vides binding sites for specific proteins containing SH2 domains, including the 85-kDa regulatory subunit of phos- phatidylinositol 3-kinase (PI3K) (13–16). Downstream from PI3K, the pleckstrin homology domain– containing serine-threonine kinase Akt is activated, and its phosphor- ylation appears to be the primary mechanism by which enzymatic activity is regulated (19). Similarly, GH pro- motes the binding of growth factor receptor bound 2 (Grb2) to IRS-1 (16,20) and Shc proteins (16,18). It was also demonstrated that GH stimulates the mitogen-acti- vated protein (MAP) kinase/ERK (21) and that one of the pathways that leads GHR to MAP kinase involves Grb2, son of Sevenless, Ras, Raf, and MAP kinase-ERK kinase (22,23). In the present study, GH-induced phosphorylation of JAK2, IRS-1, Shc, ERKs, and Akt and association of IRS-1/PI3K, IRS-1/Grb2, and Shc/Grb2 were examined in kidneys of STZ-induced diabetic rats 4 days and 8 weeks after STZ administration. In addition, the effect of G120K- PEG (a genetically engineered analog of GH produced by a mutation in the third -helix that blocks GH action, preventing GHR dimerization) on renal enlargement and GH signal transduction in kidneys of diabetic mice was evaluated. From the Department of Internal Medicine, FCM, University Of Campinas, Campinas, Sao Paulo, Brazil. Address correspondence and reprint requests to Mario J.A. Saad, Departa- mento de Clı ´nica Me ´ dica, Faculdade de Cie ˆ ncias Me ´ dicas, Universidade Estadual de Campinas, 13081-970, Campinas, SP Brasil. E-mail: msaad@fcm.unicamp.br. Received for publication 1 December 2000 and accepted in revised form 9 April 2002. J.A.S. is an employee, an officer, and a director for and holds stock in Sensus Drug Development Corporation. DAG, diacylglycerol; GH, growth hormone; GHR, growth hormone receptor; Grb2, growth factor receptor bound 2; IRS, insulin receptor substrate; MAP, mitogen-activated protein; PKB, protein kinase B; PKC, protein kinase 3; PI3K, phosphatidylinositol 3-kinase; STZ, streptozotocin. 2270 DIABETES, VOL. 51, JULY 2002 Downloaded from http://diabetesjournals.org/diabetes/article-pdf/51/7/2270/341172/db0702002270.pdf by guest on 29 October 2023