Diabetes Volume 67, September 2018 1847 Inhibition of Epidermal Growth Factor Receptor Activation Is Associated With Improved Diabetic Nephropathy and Insulin Resistance in Type 2 Diabetes Zhilian Li,1,2,3 Yan Li,2,3,4 Jessica M. Overstreet,2,3 Sungjin Chung,2,3 Aolei Niu,2,3 Xiaofeng Fan,2,3 Suwan Wang,2,3 Yinqiu Wang,2,3 Ming-Zhi Zhang,2,3 and Raymond C. Harris2,3,5 Diabetes 2018;67:1847–1857 | https://doi.org/10.2337/db17-1513 Previous studies by us and others have indicated that and decreased basal blood glucose levels. In this mouse renal epidermal growth factor receptors (EGFR) are ac- model of accelerated DN, inhibition of EGFR signaling led tivated in models of diabetic nephropathy (DN) and that to increased longevity. inhibition of EGFR activity protects against progressive DN in type 1 diabetes. In this study we examined whether inhibition of EGFR activation would affect the develop- Diabetic nephropathy (DN) is one of the most prominent PATHOPHYSIOLOGY ment of DN in a mouse model of accelerated type 2 microvascular complications of diabetes and is a major diabetes (BKS db/db with endothelial nitric oxide knock- source of morbidity and mortality. Both the incidence and 2/2 2/2 out [eNOS db/db]). eNOS db/db mice received ve- the prevalence of DN continue to rise. The vast majority hicle or erlotinib, an inhibitor of EGFR tyrosine kinase of incident and prevalent cases of DN are secondary to activity, beginning at 8 weeks of age and were sacrificed type 2 (obesity-related) diabetes. With the global rise in the at 20 weeks of age. In addition, genetic models inhibiting incidence of obesity, an increasing incidence of DN also is waved 2 a EGFR activity ( ) and transforming growth factor- being reported worldwide. waved 1 ( ) were studied in this model of DN in type 2 diabetes. The underlying mechanisms predisposing to develop- Compared with vehicle-treated mice, erlotinib-treated ment and progression of DN are an area of active investi- animals had less albuminuria and glomerulosclerosis, gation. Studies with experimental animals have identified less podocyte loss, and smaller amounts of renal a number of cytokines, hormones, and intracellular signal- profibrotic and fibrotic components. Erlotinib treat- ing pathways that may be involved in either the develop- ment decreased renal oxidative stress, macrophage and T-lymphocyte infiltration, and the production of proin- ment or the progression of DN (1). Angiotensin II and flammatory cytokines. Erlotinib treatment also pre- transforming growth factor (TGF)-b are known to play served pancreas function, and these mice had higher central roles in mediating the progressive glomerulopathy fi blood insulin levels at 20 weeks, decreased basal blood and tubulointerstitial brosis that characterize DN (2,3), glucose levels, increased glucose tolerance and insulin and renin-angiotensin-aldosterone system (RAAS) block- sensitivity, and increased blood levels of adiponectin ade is the only “specific” intervention currently available compared with vehicle-treated mice. Similar to the afore- for the treatment of patients with DN. Studies show that mentioned results, both waved 1 and waved 2 diabetic RAAS inhibition slows—but does not always prevent— mice also had attenuated DN, preserved pancreas function, progressive injury in DN (4). 1Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guang- Received 14 December 2017 and accepted 18 June 2018. dong, China This article contains Supplementary Data online at http://diabetes 2 Division of Nephrology and Hypertension, Department of Medicine, Vanderbilt .diabetesjournals.org/lookup/suppl/doi:10.2337/db17-1513/-/DC1. University School of Medicine, Nashville, TN Z.L. and Y.L. contributed equally to this work. 3Vanderbilt Center for Kidney Disease, Vanderbilt University School of Medicine, Nashville, TN © 2018 by the American Diabetes Association. Readers may use this article as 4Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of long as the work is properly cited, the use is educational and not for profit, and the Medicine, Shanghai, China work is not altered. More information is available at http://www.diabetesjournals 5Department of Veterans Affairs, Nashville, TN .org/content/license. Corresponding author: Raymond C. Harris, [email protected], or Ming-Zhi Zhang, [email protected]. 1848 EGFR and Diabetic Nephropathy Diabetes Volume 67, September 2018 Epidermal growth factor receptor (EGFR) is a member of blood samples taken from the saphenous veins of of the ErbB receptor (ErbB) family, which consists of four conscious mice at 2:00 P.M. after they had been deprived of transmembrane receptors belonging to the receptor tyro- food for 6 h, beginning at 8:00 A.M. Plasma insulin was sine kinase superfamily and includes EGFR (ErbB1/HER1), measured at the Vanderbilt Diabetes Research and Train- ErbB2/Neu/HER2, ErbB3/HER3, and ErbB4/HER4 (5–7). ing Center by using radioimmunoassay. Urinary albumin Among the four ErbB, EGFR is the prototypical receptor and creatinine excretion was determined with Albuwell M and is widely expressed in mammalian kidney, including in ELISA kits (Exocell, Philadelphia, PA). Albuminuria was the glomerulus, proximal tubule, and cortical and medul- expressed as the ratio of urinary albumin concentration to lary collecting ducts (8–11). Receptor activation leads to creatinine concentration (micrograms per milligram). phosphorylation of specific tyrosine residues within the cytoplasmic tail. These phosphorylated residues serve as Intraperitoneal Glucose Tolerance Test docking sites for a variety of signaling molecules whose Mice were deprived of food overnight (16 h), after which recruitment leads to the activation of intracellular path- a glucose solution (100 mg/mL) was injected intraperito- ways, including the mitogen-activated protein kinase, neally at a dose of 2 mg/g body wt (200 mL/10 g body wt). Janus kinase/STAT, src kinase, and phosphoinositide Then blood glucose was measured 0, 15, 30, 90, 120, and 3-kinase pathways, which control cell proliferation, differ- 150 min after glucose injection. entiation, and apoptosis (5–7). We previously reported Insulin Tolerance Test EGFR activation in experimental models of type 1 diabe- Mice were deprived of food for 6 h (8:00 A.M. to 2:00 P.M.), tesinmiceandfoundthaterlotinib,anEGFRtyrosine after which insulin was injected intraperitoneally at a dose kinase inhibitor, inhibited EGFR activation and mark- of 3 mg/kg body wt (60 mL/10 g body wt). Then blood – edly inhibited the development of DN (11 14). The study glucose was monitored 0, 15, 30, 45, 60, 75, and 90 min presented here investigated the role of EGFR signaling in after insulin administration. Insulin sensitivity was expressed mediating progressive glomerular and tubulointerstitial as a percentage of baseline blood glucose. injury in type 2 DN. We were surprised to find that inhibition of EGFR tyrosine kidney activity not only Measurement of Blood Pressure by Using a Tail Cuff retards the progression of DN but also ameliorates insulin and Carotid Catheterization resistance. Mice were anesthetized with ketamine (80 mg/g; Fort Dodge Laboratories) and inactin (8 mg/g; BYK Additives RESEARCH DESIGN AND METHODS & Instruments), administered through intraperitoneal in- jection, and placed on a temperature-controlled pad. After Animal Studies tracheostomy, phycoerythrin 10 tubing was inserted into All animal experiments were performed in accordance the right carotid artery. The catheter was tunneled under with the guidelines of the Institutional Animal Care and the skin, exteriorized, secured at the back of the neck, filled db/db UseCommitteeofVanderbiltUniversity. mice with with heparinized saline, and sealed. The catheterized mice 2/2db/db endothelial nitric oxide knockout (eNOS )on were housed individually and trained three times before a C57BLKS/J (BKS) background were generated as described blood pressure was measured with a blood pressure ana- previously (15). Genotyping was performed by PCR. lyzer (Micro-Med, Inc.) (16). Systolic blood pressure was 2/2db/db Twenty eNOS mice (8 weeks old) were treated also measured with a tail-cuff microphonic manometer daily with either the vehicle (water) or erlotinib, an EGFR (16). tyrosine kinase inhibitor, at 80 mg/kg/day, via gastric gavage for both. Mice were euthanized at 20 weeks of age. An Measurement of Plasma Adiponectin additional set of mice began treatment with the vehicle Plasma adiponectin concentration was measured using an (n =8mice)orerlotinib(n =8mice)at8weeksofageand EZMADP-60K Mouse Adiponectin ELISA Kit (Millipore), continued treatment until 40 weeks of age or death. Both according to the manufacturer’s instructions. waved 1 mice harboring a null mutation of TGF-a, an EGFR Determination of Urinary F2-Isoprostane ligand (stock no. 000004), and waved 2 mice harboring Mice were trained three times in metabolic cages (Brain- a point mutation in the EGFR gene, with resultant loss tree Scientific, Inc., Braintree, MA) before urine was of most of its tyrosine kinase activity (stock no. 025148), collected continuously for 16 h. A single mouse was were purchased from The Jackson Laboratory. These mice 2 2 placed in a metabolic cage overnight and then returned to were crossed with eNOS / db/+ mice to eventually get 2 2 2 2 its original cage for 2 days before the next training period. wa1/+;eNOS / db/db (control) and wa1/wa1;eNOS / db/db 2 2 The metabolic cages were humidified to minimize the (waved 1) mice, as well as wa2/+;eNOS / db/db (control) 2 2 evaporation of the urine samples when 16-h urine samples and wa2/wa2;eNOS / db/db (waved 2)mice. were collected. F2-isoprostanes, which are prostaglandin Measurements of Blood Glucose, Insulin, and Urinary F2–like compounds, have been recognized as sensitive and Albumin Excretion specific biomarkers of oxidative stress. Urinary F2-isoprostane Fasting blood glucose was evaluated with a B-Glucose levels were determined by negative-ion gas chromatography– Analyzer (HemoCue, Lake Forest, CA) through the use mass spectrometry (17).