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ASK1 Inhibitor Halts Progression of Diabetic Nephropathy in Nos3-Deficient Mice

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ASK1 Inhibitor Halts Progression of Diabetic Nephropathy in Nos3-Deficient Mice Diabetes Volume 64, November 2015 3903 Greg H. Tesch,1,2 Frank Y. Ma,1,2 Yingjie Han,1,2 John T. Liles,3 David G. Breckenridge,3 and David J. Nikolic-Paterson1,2 ASK1 Inhibitor Halts Progression of Diabetic Nephropathy in Nos3-Deficient Mice Diabetes 2015;64:3903–3913 | DOI: 10.2337/db15-0384 p38 mitogen-activated protein kinase (MAPK) signaling end-stage disease (1), indicating a major gap in our treat- promotes diabetic kidney injury. Apoptosis signal- ment options. The diabetic kidney is stressed by multiple regulating kinase (ASK)1 is one of the upstream kinases factors including hyperglycemia, reactive oxygen species, in the p38 MAPK-signaling pathway, which is activated by advanced glycation end products, proinflammatory cyto- inflammation and oxidative stress, suggesting a possible kines, and angiotensin II—all of which can induce signaling role for ASK1 in diabetic nephropathy. In this study, we via the p38 mitogen-activated protein kinase (MAPK) examined whether a selective ASK1 inhibitor can pre- – (2 5). Increased activation of p38 MAPK in glomeruli and COMPLICATIONS vent the induction and progression of diabetic nephrop- the tubulointerstitial compartment has been described in athy in mice. Diabetes was induced in hypertensive animal models of diabetic nephropathyaswellasinpatients Nos3 fi endothelial nitric oxide synthase ( )-de cient mice with diabetic nephropathy (6–9). Inhibition of p38 MAPK fi by ve low-dose streptozotocin (STZ) injections. Groups activation via pharmacologic and genetic approaches can of diabetic Nos32/2 mice received ASK1 inhibitor (GS- suppress the induction of albuminuria, glomerular matrix 444217 delivered in chow) as an early intervention (2–8 expansion, and inflammation in models of type 1 and type weeks after STZ) or late intervention (weeks 8–15 after 2/2 2 diabetic nephropathy (9,10), although intervention STZ). Control diabetic and nondiabetic Nos3 mice studies in established disease are lacking. Importantly, received normal chow. Treatment with GS-444217 abro- gated p38 MAPK activation in diabetic kidneys but had clinical trials of p38 inhibitor compounds in rheumatoid no effect upon hypertension in Nos32/2 mice. Early arthritis have failed to deliver the promise of animal stud- intervention with GS-444217 significantly inhibited dia- ies owing to toxicity issues, which have led to investigation of betic glomerulosclerosis and reduced renal dysfunction the upstream kinases involved in context-dependent p38 but had no effect on the development of albuminuria. MAPK activation (11). Late intervention with GS-444217 improved renal func- Apoptosis signal-regulating kinase 1 (ASK1/MAP3K5) tion and halted the progression of glomerulosclerosis, is a member of the large family of MAPK kinase kinase renal inflammation, and tubular injury despite having no enzymes, many of which have the potential to activate the effect on established albuminuria. In conclusion, this downstream p38 MAPK via phosphorylation of the MKK3 study identifies ASK1 as a new therapeutic target in and MKK6 enzymes (12,13). p38 MAPK and, to a lesser diabetic nephropathy to reduce renal inflammation and extent, c-Jun terminal kinase are the only known down- fibrosis independent of blood pressure control. stream targets of ASK1 signaling (14). ASK1 is activated in response to oxidative stress; specifically, ASK1 exists as an inactive dimer coupled to thioredoxin and undergoes Diabetic nephropathy is the most common single cause of autoactivation after the oxidation and dissociation of thi- end-stage renal failure in many countries. Current ther- oredoxin (13). ASK1 is widely expressed in diverse tissues apies of controlling blood pressure and blood glucose and is most highly expressed in the kidney (15). Mice 2/2 levels have only a limited benefit in slowing progression to lacking the Ask1 gene (Ask1 ) have a normal phenotype, 1Department of Nephrology, Monash Medical Centre, Clayton, Victoria, Australia This article contains Supplementary Data online at http://diabetes 2Monash University Department of Medicine, Clayton, Victoria, Australia .diabetesjournals.org/lookup/suppl/doi:10.2337/db15-0384/-/DC1. 3 Gilead Sciences, Foster City, CA © 2015 by the American Diabetes Association. Readers may use this article as Corresponding author: Greg H. Tesch, [email protected]. long as the work is properly cited, the use is educational and not for profit, and Received 19 March 2015 and accepted 6 July 2015. the work is not altered. 3904 ASK1 in Diabetic Nephropathy Diabetes Volume 64, November 2015 including normal kidney structure and function, which con- the mice in all studies, and no drug-related clinical obser- trasts with the fetal lethality of mice lacking the p38a/ vations were noted in any of the studies. Blood glucose 2/2 Mapk14 gene (16,17). In addition, Ask1 mice show a dra- (measured by a tail vein sample) and body weight were matic inhibition of p38 MAPK activation and significant measured weekly after a 3-h fast (0800–1100 h), and mice protection from kidney injury in models of acute tubular with fasting glucose levels .30 mmol/L were given 0.5 necrosis and renal interstitial fibrosis, providing results units s.c. protophane insulin (Novo Nordisk, Sydney, Aus- consistent with administration of p38 inhibitors (4,18–20). tralia) three times a week to maintain body weight. Urine Therefore, inhibition of ASK1/p38 signaling may have ther- was collected pre-experiment and at weeks 5, 8, 12, and apeutic potential for diabetic glomerulosclerosis. 15 after STZ injection to assess urine albumin excretion. This aim of this study was to investigate the therapeutic Glycated hemoglobin (HbA1c) was measured from blood potential of a highly selective small molecule ASK1 in- samples taken at weeks 8 and 15. Tissues were collected at hibitor (GS-444217) in experimental diabetic nephropathy. week 8 or 15 and were fixed in 4% (v/v) formaldehyde or We investigated streptozotocin (STZ)-induced type 1 di- 2% (w/v) paraformaldehyde-lysine-periodate or snap- abetes in hypertensive mice lacking the gene for endothe- frozenandstoredat280°C. These animal studies were lial nitric oxide synthase (Nos3), as this model exhibits approved by the Monash Medical Centre Animal Ethics robust development of diabetic glomerulosclerosis (21). Committee in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific RESEARCH DESIGN AND METHODS Purposes, 7th edition (2004). ASK1 Inhibitor Biochemistry GS-444217 was synthesized by Gilead Sciences in Foster Fasting blood glucose was measured from tail blood by City, CA (manuscript in preparation). In a competitive, glucometer (Medisense, Abbott Laboratories, Bedford, fl time-resolved uorescence resonance energy-transfer im- MA). Urine was collected from mice housed in metabolic munoassay, GS-444217 directly inhibited ASK1 kinase cages for 8 h. Heparinized whole blood was collected from activity in vitro with an IC50 of 2.9 6 0.8 nmol/L. In tail veins for analysis of HbA1c. At the end of experimen- a kinase selectivity panel that included 451 kinases tation, whole blood was collected by cardiac puncture in (KINOMEscan; DiscoverRX Corporation, Fremont, CA), anesthetized animals and stored as serum or heparinized . fi GS-444217 exhibited 50-fold greater af nity for ASK1 plasma. Urine creatinine levels were determined by the compared with all other kinases measured. For delivery in Jaffe rate reaction method. ELISA kits were used to assess mice, GS-444217 was incorporated into standard mouse levels of urine albumin (Bethyl Laboratories, Montgom- chow at 0.1% (w/w). The concentration of GS-444217 in ery, TX), plasma insulin (Mercodia, Uppsala, Sweden), and mouse plasma was measured at the end of each study and serum cystatin-C (Enzo Life Sciences, Farmingdale, NY). 6 m found to be 20.2 8.2 mol/L for the early-intervention HbA was measured by DCA Vantage Analyzer (Siemens, 6 m 1c study and 21.3 7.4 mol/L for the late-intervention Camberley, U.K.). study. These exposures of GS-444217 in mice are expected to result in complete and selective inhibition Blood Pressure Analysis of ASK1 (data not shown). Systolic blood pressure was measured in conscious mice by tail-cuff plethysmography (IITC Life Science, Woodland Animal Model of Diabetic Nephropathy Hills, CA). Mice were trained twice weekly for 3 weeks 2/2 Nos3 mice on the C57BL/6 background were obtained prior to experimental readings. At each recording, mice from The Jackson Laboratory (Bar Harbor, ME) and bred were acclimatized to a preheated chamber (29°C) for 15 under pathogen-free conditions at the Monash Medical min and the pressure readings were recorded over three Centre Animal Facility (Clayton, Australia). At 8 weeks consecutive manual inflation-deflation cycles to obtain an 2/2 of age, male Nos3 mice were given injections of STZ average. (5 3 55 mg/kg/day i.p.; Sigma, St Louis, MO). Groups of Antibodies mice (n = 10) with diabetes (fasting blood glucose .16 The following primary antibodies were used in this study: mmol/L at 2 weeks after the last STZ injection) were mouse anti–phospho-p38a (Upstate Biotechnology, Lake followed for 8 or 15 weeks. In the early-intervention Placid, NY), rat anti-CD68 (FA-11; Serotec, Oxford, U.K.), study, diabetic mice were fed either normal chow or goat anti–collagen IV (Southern Biotechnology, Birming- chow containing 0.1% GS-444217 between weeks 2 and ham, AL), and rabbit anti–Wilms tumor 1 (WT1) antigen 8 and then killed. In the late-intervention study, diabetic (Santa Cruz Biotechnology, Santa Cruz, CA). mice were fed either normal chow or chow with 0.1% GS- 444217 over weeks 8–15 and then killed. Age-matched Immunohistochemistry 2/2 nondiabetic Nos3 mice (n = 10) were used as controls. Formalin-fixed sections (2 mm) were stained with periodic 2/2 In an additional study, a group of nondiabetic Nos3 acid-Schiff (PAS) to assess structure and counterstained mice (n = 9) was assessed for systolic blood pressure be- with hematoxylin to identify nuclei.
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