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 Progressionof Diabetic Nephropathy inNos3-Deficient MiceDiabetes 2015;64:3903–3913 | DOI: 10.2337/db15-0384

p38 mitogen-activated protein kinase (MAPK) signalingpromotes diabetic kidney injury. Apoptosis signal-regulating kinase (ASK)1 is one of the upstream kinasesin the p38 MAPK-signaling pathway, which is activated byinflammation and oxidative stress, suggesting a possiblerole for ASK1 in diabetic nephropathy. In this study, weexamined whether a selective ASK1 inhibitor can pre-vent the induction and progression of diabetic nephrop-athy in mice. Diabetes was induced in hypertensiveendothelial nitric oxide synthase (Nos3)-deficient miceby five low-dose streptozotocin (STZ) injections. Groupsof diabetic Nos32/2 mice received ASK1 inhibitor (GS-444217 delivered in chow) as an early intervention (2–8weeks after STZ) or late intervention (weeks 8–15 afterSTZ). Control diabetic and nondiabetic Nos32/2 micereceived normal chow. Treatment with GS-444217 abro-gated p38 MAPK activation in diabetic kidneys but hadno effect upon hypertension in Nos32/2 mice. Earlyintervention with GS-444217 significantly inhibited dia-betic glomerulosclerosis and reduced renal dysfunctionbut had no effect on the development of albuminuria.Late intervention with GS-444217 improved renal func-tion and halted the progression of glomerulosclerosis,renal inflammation, and tubular injury despite having noeffect on established albuminuria. In conclusion, thisstudy identifies ASK1 as a new therapeutic target indiabetic nephropathy to reduce renal inflammation andfibrosis independent of blood pressure control.

Diabetic nephropathy is the most common single cause ofend-stage renal failure in many countries. Current ther-apies of controlling blood pressure and blood glucoselevels have only a limited benefit in slowing progression to

end-stage disease (1), indicating a major gap in our treat-ment options. The diabetic kidney is stressed by multiplefactors including hyperglycemia, reactive oxygen species,advanced glycation end products, proinflammatory cyto-kines, and angiotensin II—all of which can induce signalingvia the p38 mitogen-activated protein kinase (MAPK)(2–5). Increased activation of p38 MAPK in glomeruli andthe tubulointerstitial compartment has been described inanimal models of diabetic nephropathy as well as in patientswith diabetic nephropathy (6–9). Inhibition of p38 MAPKactivation via pharmacologic and genetic approaches cansuppress the induction of albuminuria, glomerular matrixexpansion, and inflammation in models of type 1 and type2 diabetic nephropathy (9,10), although interventionstudies in established disease are lacking. Importantly,clinical trials of p38 inhibitor compounds in rheumatoidarthritis have failed to deliver the promise of animal stud-ies owing to toxicity issues, which have led to investigation ofthe upstream kinases involved in context-dependent p38MAPK activation (11).

Apoptosis signal-regulating kinase 1 (ASK1/MAP3K5)is a member of the large family of MAPK kinase kinaseenzymes, many of which have the potential to activate thedownstream p38 MAPK via phosphorylation of the MKK3and MKK6 enzymes (12,13). p38 MAPK and, to a lesserextent, c-Jun terminal kinase are the only known down-stream targets of ASK1 signaling (14). ASK1 is activatedin response to oxidative stress; specifically, ASK1 exists asan inactive dimer coupled to thioredoxin and undergoesautoactivation after the oxidation and dissociation of thi-oredoxin (13). ASK1 is widely expressed in diverse tissuesand is most highly expressed in the kidney (15). Micelacking the Ask1 gene (Ask12/2) have a normal phenotype,

1Department of Nephrology, Monash Medical Centre, Clayton, Victoria, Australia2Monash University Department of Medicine, Clayton, Victoria, Australia3Gilead Sciences, Foster City, CA

Corresponding author: Greg H. Tesch, greg.tesch@monash.edu.

Received 19 March 2015 and accepted 6 July 2015.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db15-0384/-/DC1.

© 2015 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

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including normal kidney structure and function, which con-trasts with the fetal lethality of mice lacking the p38a/Mapk14 gene (16,17). In addition, Ask12/2mice show a dra-matic inhibition of p38 MAPK activation and significantprotection from kidney injury in models of acute tubularnecrosis and renal interstitial fibrosis, providing resultsconsistent with administration of p38 inhibitors (4,18–20).Therefore, inhibition of ASK1/p38 signaling may have ther-apeutic potential for diabetic glomerulosclerosis.

This aim of this study was to investigate the therapeuticpotential of a highly selective small molecule ASK1 in-hibitor (GS-444217) in experimental diabetic nephropathy.We investigated streptozotocin (STZ)-induced type 1 di-abetes in hypertensive mice lacking the gene for endothe-lial nitric oxide synthase (Nos3), as this model exhibitsrobust development of diabetic glomerulosclerosis (21).

RESEARCH DESIGN AND METHODS

ASK1 InhibitorGS-444217 was synthesized by Gilead Sciences in FosterCity, CA (manuscript in preparation). In a competitive,time-resolved fluorescence resonance energy-transfer im-munoassay, GS-444217 directly inhibited ASK1 kinaseactivity in vitro with an IC50 of 2.9 6 0.8 nmol/L. Ina kinase selectivity panel that included 451 kinases(KINOMEscan; DiscoverRX Corporation, Fremont, CA),GS-444217 exhibited .50-fold greater affinity for ASK1compared with all other kinases measured. For delivery inmice, GS-444217 was incorporated into standard mousechow at 0.1% (w/w). The concentration of GS-444217 inmouse plasma was measured at the end of each study andfound to be 20.2 6 8.2 mmol/L for the early-interventionstudy and 21.3 6 7.4 mmol/L for the late-interventionstudy. These exposures of GS-444217 in mice areexpected to result in complete and selective inhibitionof ASK1 (data not shown).

Animal Model of Diabetic NephropathyNos32/2 mice on the C57BL/6 background were obtainedfrom The Jackson Laboratory (Bar Harbor, ME) and bredunder pathogen-free conditions at the Monash MedicalCentre Animal Facility (Clayton, Australia). At 8 weeksof age, male Nos32/2 mice were given injections of STZ(5 3 55 mg/kg/day i.p.; Sigma, St Louis, MO). Groups ofmice (n = 10) with diabetes (fasting blood glucose .16mmol/L at 2 weeks after the last STZ injection) werefollowed for 8 or 15 weeks. In the early-interventionstudy, diabetic mice were fed either normal chow orchow containing 0.1% GS-444217 between weeks 2 and8 and then killed. In the late-intervention study, diabeticmice were fed either normal chow or chow with 0.1% GS-444217 over weeks 8–15 and then killed. Age-matchednondiabetic Nos32/2 mice (n = 10) were used as controls.In an additional study, a group of nondiabetic Nos32/2

mice (n = 9) was assessed for systolic blood pressure be-fore and after a 6-week period on chow containing 0.1%GS-444217. GS-444217 treatment was well tolerated by

the mice in all studies, and no drug-related clinical obser-vations were noted in any of the studies. Blood glucose(measured by a tail vein sample) and body weight weremeasured weekly after a 3-h fast (0800–1100 h), and micewith fasting glucose levels .30 mmol/L were given 0.5units s.c. protophane insulin (Novo Nordisk, Sydney, Aus-tralia) three times a week to maintain body weight. Urinewas collected pre-experiment and at weeks 5, 8, 12, and15 after STZ injection to assess urine albumin excretion.Glycated hemoglobin (HbA1c) was measured from bloodsamples taken at weeks 8 and 15. Tissues were collected atweek 8 or 15 and were fixed in 4% (v/v) formaldehyde or2% (w/v) paraformaldehyde-lysine-periodate or snap-frozen and stored at 280°C. These animal studies wereapproved by the Monash Medical Centre Animal EthicsCommittee in accordance with the Australian Code ofPractice for the Care and Use of Animals for ScientificPurposes, 7th edition (2004).

BiochemistryFasting blood glucose was measured from tail blood byglucometer (Medisense, Abbott Laboratories, Bedford,MA). Urine was collected from mice housed in metaboliccages for 8 h. Heparinized whole blood was collected fromtail veins for analysis of HbA1c. At the end of experimen-tation, whole blood was collected by cardiac puncture inanesthetized animals and stored as serum or heparinizedplasma. Urine creatinine levels were determined by theJaffe rate reaction method. ELISA kits were used to assesslevels of urine albumin (Bethyl Laboratories, Montgom-ery, TX), plasma insulin (Mercodia, Uppsala, Sweden), andserum cystatin-C (Enzo Life Sciences, Farmingdale, NY).HbA1c was measured by DCA Vantage Analyzer (Siemens,Camberley, U.K.).

Blood Pressure AnalysisSystolic blood pressure was measured in conscious miceby tail-cuff plethysmography (IITC Life Science, WoodlandHills, CA). Mice were trained twice weekly for 3 weeksprior to experimental readings. At each recording, micewere acclimatized to a preheated chamber (29°C) for 15min and the pressure readings were recorded over threeconsecutive manual inflation-deflation cycles to obtain anaverage.

AntibodiesThe following primary antibodies were used in this study:mouse anti–phospho-p38a (Upstate Biotechnology, LakePlacid, NY), rat anti-CD68 (FA-11; Serotec, Oxford, U.K.),goat anti–collagen IV (Southern Biotechnology, Birming-ham, AL), and rabbit anti–Wilms tumor 1 (WT1) antigen(Santa Cruz Biotechnology, Santa Cruz, CA).

ImmunohistochemistryFormalin-fixed sections (2 mm) were stained with periodicacid-Schiff (PAS) to assess structure and counterstainedwith hematoxylin to identify nuclei. Immunostaining forphospho-p38, WT1, and collagen IV was performed on4 mm paraffin-embedded sections, which were fixed in 4%

3904 ASK1 in Diabetic Nephropathy Diabetes Volume 64, November 2015

formalin or methylcarn solution. Immunostaining forCD68 was performed on 5 mm paraformaldehyde-lysine-periodate–fixed cryostat sections. For antigen retrieval ofphospho-p38, dewaxed paraffin sections were heated ina microwave oven (800 watts for 12 min) in 10 mmol/Lsodium citrate buffer (pH 6.0) (22). For immunostaining,sections were treated with 20% rabbit serum or 20% goatserum for 30 min and then incubated with primary anti-body in 3% BSA overnight at 4°C. Sections were thenplaced in 0.6% hydrogen peroxide in methanol for 20min to inactivate endogenous peroxidase. Bound primaryantibodies were detected using a standard ABC-peroxidasesystem: avidin-biotin block, biotinylated antibodies (rabbitanti-goat IgG, rabbit anti-rat IgG, or goat anti-mouse IgG),and ABC-peroxidase (Vector Laboratories, Burlingame, CA).Sections were developed with 3,3-diaminobenzidine (Sigma)to produce a brown color. CD68 sections were counter-stained with hematoxylin to assist cell counting. Normalgoat serum or isotype-matched irrelevant IgG was used asnegative control.

Quantitation of ImmunohistochemistryThe number of CD68+ cells and WT1+ podocytes wascounted in 30 hilar glomerular cross-sections (gcs) peranimal (3400). Glomerular collagen IV staining was quan-titated by computer image analysis (Image-Pro Plus,Media Cybernetics, Silver Spring, MD) in 50 hilar glomer-ular cross-sections (3400) and expressed as the percent-age of glomerular area stained. All scoring was performedon blinded slides.

Real-time PCRTotal RNA was extracted from whole kidney using Trizol(Invitrogen) and reverse transcribed with random primersusing the Superscript First-Strand Synthesis kit (Invitrogen).Real-time PCR analysis was performed as previouslydescribed (5,23). The Taqman probe and primer sequencesare listed in Supplementary Table 1.

Statistical AnalysisStatistical differences were analyzed by one-way ANOVAwith Tukey multiple comparison posttest. Data wererecorded as mean 6 SEM with P , 0.05 considered sig-nificant. All analyses were performed using GraphPadPrism 6.0 (GraphPad Software, San Diego, CA).

RESULTS

Effect of ASK1 Inhibitor on p38 MAPK Signaling andBlood PressureConsistent with previous studies (6,9,24), basal p38 phos-phorylation (activation) was detected in nondiabetic kid-ney by Western blot, while immunostaining identifiedphosphorylated p38 in glomeruli, tubules, and the inter-stitium (Fig. 1A and E). A significant increase in p38 ac-tivation in the kidney was evident at both weeks 8 and15 (Fig. 1). In contrast, treatment with GS-444217 overweeks 2–8 and over weeks 8–15 of diabetes caused a dra-matic inhibition of p38 activation in all cell types (Fig. 1).

Since diabetes does not affect hypertension in Nos32/2

mice (25,26), we measured the effect of GS-444217 onblood pressure in nondiabetic Nos32/2 mice. Systolicblood pressure in Nos32/2 mice was 133 6 5 mmHgbefore treatment and was unchanged (134 6 5 mmHg)after 6 weeks of GS-444217 administration, demonstrat-ing that GS-444217 does not affect hypertension inthese mice.

Early Intervention With GS-444217 Inhibits theDevelopment of Diabetic Renal InjuryGroups of diabetic Nos32/2 mice were fed standard chowor chow with 0.1% GS-444217 from week 2 after STZinjection until being killed on week 8. GS-444217 hadno effect upon fasting body weight, fasting blood glucoselevels, or plasma insulin levels, although a partial reduc-tion in HbA1c levels was evident in the drug-treated group(Fig. 2). A substantial increase in urinary albumin excre-tion was evident on weeks 5 and 8 after STZ injection,which was not affected by GS-444217 treatment (Fig. 3A).There was also a reduction in the number of glomerularWT1+ podocytes at week 8 after STZ, which was unaf-fected by drug treatment (Fig. 3B). However, the declinein renal function at week 8 after STZ, as assessed by in-creased serum cystatin C levels, was significantly im-proved by drug treatment (Fig. 3C).

Nos32/2 mice with no treatment developed mild-to-moderate glomerulosclerosis by week 8 of diabetes, asshown by increased PAS-stained glomerular depositsand an increase in glomerular staining for collagen IV(Fig. 4). In addition, a significant glomerular infiltrate ofCD68+ macrophages was evident (Fig. 4). GS-444217 sig-nificantly reduced both glomerulosclerosis and glomerularmacrophage infiltration (Fig. 4).

Late Intervention With GS-444217 Improves RenalFunction and Halts Progressive Glomerulosclerosis inEstablished Diabetic NephropathyGroups of diabetic Nos32/2 mice were fed standard chowor chow with 0.1% GS-444217 from week 8 after STZinjection until being killed on week 15. In animals fedthe standard diet, disease severity was maintained or in-creased between weeks 8 and 15 (Figs. 5 and 6). GS-444217 had no effect upon fasting body weight, fastingblood glucose levels, or HbA1c levels (Fig. 5). The diabeticmice had established albuminuria and raised serumcystatin-C levels at week 8, when GS-444217 treatmentcommenced (Fig. 6). Drug treatment had no impact uponestablished albuminuria or podocyte loss, but there wasa significant reduction in the levels of serum cystatin-C,suggesting that GS-444217 treatment improved renalfunction (Fig. 6).

There was a clear increase in diabetic glomeruloscle-rosis between weeks 8 and 15 in untreated diabeticNos32/2 mice as shown by glomerular collagen IV stain-ing (Fig. 7A–C and E). This was associated with a sustainedincrease in renal mRNA levels for profibrotic molecules(collagens 1 and 4, fibronectin, transforming growth

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factor-b1, and plasminogen activator inhibitor-I) (Fig. 7F–J)and a sustained glomerular CD68+ macrophage infiltrateand increases in renal mRNA levels for proinflammatorymolecules (chemokine CC motif ligand 2 [CCL2] and tumor

necrosis factor-a) (Fig. 8A–F). In addition, expression ofthe tubular damage marker kidney injury molecule-1(KIM-1) was elevated at week 8 of diabetes and remainedelevated through to week 15 (Fig. 8G).

Figure 1—Effect of ASK1 inhibitor (ASK1i) GS-444217 on p38 MAPK activation in the diabetic kidney. Western blot analysis of phosphor-ylated (p)-p38 in whole kidney samples (A) and quantification of phosphorylated p38 compared with the tubulin control (B) in the early-intervention study. Groups of diabetic Nos32/2 mice were fed standard chow (no treatment [No Tx]) or chow with 0.1% GS-444217 fromweek 2 after STZ injection until being killed at week 8. Age-matched nondiabetic Nos32/2 mice were used as controls (No STZ). Westernblot analysis (C) and quantification (D) of p-p38 in whole kidney samples in the late-intervention study. Groups of diabetic Nos32/2 micewere fed standard chow (No Tx) or chow with 0.1% GS-444217 from week 8 after STZ injection until being killed at week 15. Age-matchednondiabetic Nos32/2 mice were used as controls (No STZ). Kidney immunostaining for phosphorylated p38 (brown) at week 8 (E–G) andweek 15 (H–J) of diabetes. Immunostaining for phosphorylated p38 was seen in glomeruli, tubules, and the interstitium of nondiabeticNos32/2 control kidneys at week 8 (E ) and week 15 (H). An increase in p-p38 was evident in diabetic kidneys, particularly within glomeruliand dilated tubules at week 8 (F ), and was similarly elevated at week 15 (I). GS-444217 treatment largely abrogated the phosphorylated p38staining in diabetic kidneys at week 8 (G) and week 15 (J). Original magnification (E–G) 3250. ***P < 0.001. w, weeks.

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GS-444217 treatment over weeks 8–15 preventedthe increase in glomerular deposition of collagen IV anddownregulated renal mRNA levels for all of the profibroticmolecules analyzed (Fig. 7D–J). In addition, GS-444217treatment significantly reduced the glomerular macro-phage infiltrate and reduced the renal mRNA expressionof proinflammatory markers and tubular damage markerKIM-1 (Fig. 8).

DISCUSSION

Administration of an ASK1 inhibitor, GS-444217, wasshown to be effective in a mouse model of establisheddiabetic nephropathy on the basis of halting progressiveglomerulosclerosis, reducing inflammation, and improv-ing renal function despite ongoing hyperglycemia, hyper-tension, and albuminuria.

The protective effect of ASK1 inhibitor treatment indiabetic nephropathy was attributed to the highly effec-tive blockade of p38 activation—the major downstreamtarget for ASK1 (14). GS-444217 is highly selective forASK1, although as in all pharmacology studies, we cannotformally rule out unanticipated off-target effects. In-creased p38 activation has been described in human andexperimental diabetic nephropathy (6–9), which was con-firmed in the current study. Administration of a p38MAPK inhibitor has been shown to reduce albuminuriaand prevent the development of mild glomerular fibrosisin nonhypertensive diabetic rats (10). In addition, dele-tion of the Mkk3 gene in db/db mice with type 2 diabetes

significantly reduced p38 activation, albuminuria, podocyteloss, and glomerular collagen deposition and improved re-nal function (9). The current study significantly extendsthese findings in two ways. First, diabetic Nos32/2 micedevelop a much more severe form of glomerulosclerosiscompared with the above two models (21), thereby pro-viding a much sterner test of the ability of ASK1/p38blockade to prevent diabetic glomerulosclerosis. Second,we performed an intervention study and demonstratedthat GS-444217 could inhibit p38 activation, halt glomer-ulosclerosis, reduce glomerular inflammation, and improverenal function in established diabetic nephropathy. Indeed,the results of the current study are an important compo-nent of the preclinical data that support a current phase 2

Figure 2—Effect of ASK1 inhibitor (ASK1i) GS-444217 on the de-velopment of diabetes in Nos32/2 mice. Groups of diabetic Nos32/2

mice were fed standard chow (no treatment [No Tx]) or chow with0.1% GS-444217 from week 2 after STZ injection until being killedon week 8. Age-matched nondiabetic Nos32/2 mice were used ascontrols (No STZ). Fasting body weight (A), fasting blood glucose(FBG) (B), HbA1c levels (C), and plasma insulin levels (D). NS, notsignificant. ***P < 0.001.

Figure 3—Effect of ASK1 inhibitor (ASK1i) GS-444217 on albumin-uria, podocyte loss, and renal function in early diabetes. Groupsof diabetic Nos32/2 mice were fed standard chow (no treatment[No Tx]) or chow with 0.1% GS-444217 from week 2 after STZ in-jection until being killed on week 8. Age-matched nondiabetic Nos32/2

mice were used as controls (No STZ). Urine albumin-to-creatinine ratio(ACR) (A), number of WT1+ podocytes per glomerular cross-section(B), and renal function measured by serum cystatin-C levels (C). NS,not significant. **P < 0.05, ***P < 0.001.

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trial evaluating an ASK1 inhibitor, GS-4997, in patientswith stage 3/4 diabetic kidney disease (27).

The ability of GS-444217 to reduce collagen depositionin the diabetic glomerulus is consistent with previousstudies in which p38 inhibitors have been shown toreduce renal fibrosis in nondiabetic models of kidneydisease (19,28,29). In addition, Ask12/2 mice show a pro-found suppression of p38 activation and tissue fibrosis inmodels of both renal and cardiac disease (4,30,31).

A second mechanism whereby ASK1 inhibition reduceddiabetic glomerular damage is through the inhibition ofmacrophage infiltration and inhibition of mRNA levels ofproinflammatory mediators. Previous studies have shownthat macrophages cause glomerular injury in experimentmodels of diabetic nephropathy (32,33). This is consistentwith the reduction in CCL2 mRNA levels and macrophageinfiltration seen in the tubulointerstitium in the obstructedkidney in Ask12/2 mice (4).

Figure 4—Effect of ASK1 inhibitor (ASK1i) GS-444217 on glomerular damage in early diabetes. A–C: PAS-stained kidney sections.A: Nondiabetic control Nos32/2 kidney. B: Significant PAS-stained deposits were seen in glomeruli after 8 weeks of diabetes in micewith no treatment. C: Treatment with GS-444217 over weeks 2–8 reduced glomerular PAS-stained deposits. D–F: Immunostaining forcollagen IV (brown). D: Nondiabetic control Nos32/2 kidney showing staining of collagen IV in the glomerular basement membrane. E:Significant mesangial deposition of collagen IV after 8 weeks of diabetes with no treatment. F: Treatment with GS-444217 suppressedglomerular collagen IV deposition. G–I: Immunostaining for CD68+ macrophages (brown) with weak hematoxylin and PAS staining. G: Non-diabetic control Nos32/2 kidney showing a single CD68+ cell in a glomerulus. H: Increased numbers of glomerular CD68+ cells were seen inuntreated mice after 8 weeks of diabetes. I: Treatment with GS-44217 reduced the number of glomerular CD68+ cells in diabetic mice. J: Imageanalysis of the area of glomerular collagen IV staining (glom area). K: Quantification of the number of glomerular CD68+ macrophages. Originalmagnification (A–I) 3400. *P < 0.05, **P < 0.01, ***P < 0.001.

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The aggressive nature of disease development indiabetic Nos32/2 mice is attributed to the presence ofhypertension. However, the ASK1 inhibitor protectedagainst diabetic glomerulosclerosis despite having no ef-fect on hypertension in Nos32/2 mice. This is consistentwith previous studies on Ask12/2 mice, which are pro-tected from end organ damage in the setting of hyperten-sion. For example, in models of angiotensin II andmineralocorticoid-driven hypertension, Ask12/2 micehad reduced cardiac fibrosis despite unaltered hyperten-sion (30,31). In the current study, ASK1 inhibition withGS-444217 appears to reduce renal injury and improverenal function by directly decreasing inflammation andfibrosis in the diabetic kidney.

The loss of podocytes and development of albuminuriain diabetic Nos32/2 mice were unaffected by ASK1 inhib-itor treatment in our early- and late-intervention studies.This suggests that activation of ASK1/p38 signaling is notinvolved in the mechanism of diabetic albuminuria. How-ever, there are some limitations in this interpretation.Recent studies of Nos32/2 mice has shown that theyare highly susceptible to glucose-induced podocyte damage

Figure 5—Effect of ASK1 inhibitor (ASK1i) GS-444217 on the pro-gression of diabetes in Nos32/2 mice. Groups of diabetic Nos32/2

mice were fed standard chow (no treatment [No Tx]) or chow with0.1% GS-444217 from week 8 after STZ injection until being killedon week 15. Age-matched nondiabetic Nos32/2 mice were used ascontrols (No STZ). Fasting body weight (FBW) (A), fasting bloodglucose (FBG) (B), and HbA1c levels (C).

Figure 6—Effect of ASK1 inhibitor (ASK1i) GS-444217 on albumin-uria, podocyte loss, and renal function in progressive diabetes.Groups of diabetic Nos32/2 mice were fed standard chow (no treat-ment [No Tx]) or chow with 0.1% GS-444217 from week 8 after STZinjection until being killed on week 15. Age-matched nondiabeticNos32/2 mice were used as controls (No STZ). Urine albumin-to-creatinine ratio (ACR) (A), number of WT1+ podocytes per glomerularcross-section (B), and renal function measured by serum cystatin-Clevels (C). NS, not significant; wk, week. ***P < 0.001 vs. STZ-No Tx.

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Figure 7—Effect of ASK1 inhibitor (ASK1i) GS-444217 on kidney fibrosis in progressive diabetes. A–D: Immunostaining for collagen IV.A: Nondiabetic control Nos32/2 kidney. B: Significant mesangial deposition of collagen (Col) IV after 8 weeks of diabetes with no treatment.C: Enhanced glomerular collagen IV deposition after 15 weeks of diabetes with no treatment. D: GS-444217 treatment over weeks 8–15prevents further glomerular collagen IV deposition. E: Image analysis of the area of glomerular collagen IV staining (glom area). F–J: Real-time PCR analysis of whole kidney mRNA levels for collagen IV (F ), collagen I (G), fibronectin (FN) (H), transforming growth factor-b1 (TGF-b1) (I), and plasminogen activator inhibitor-1 (PAI-1) (J). Original magnification (A–D) 3400. NS, not significant. No Tx, no treatment; NoSTZ, controls. *P < 0.05, **P < 0.01, ***P < 0.001.

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and show marked albuminuria 2 weeks after STZ admin-istration (34), which was the time at which we beganASK1 inhibitor treatment in the early-intervention study.Therefore, it is possible that ASK1 inhibition may havebeen commenced too late to have an effect on the induc-tion of podocyte damage/loss and albuminuria. Previous

studies in both diabetic and nondiabetic kidney diseasehave shown that p38 inhibitors can reduce albuminuria(9,10,28,35), arguing that ASK1/p38 signaling may con-tribute to albuminuria in some contexts.

Early intervention with the ASK1 inhibitor saw a re-duction in HbA1c levels in diabetic Nos32/2 mice despite

Figure 8—Effect of ASK1 inhibitor (ASK1i) GS-444217 on kidney inflammation and tubular damage in progressive diabetes. A and B:Immunostaining for CD68+ macrophages (brown) with weak hematoxylin and PAS staining. A: A Nos32/2 kidney at week 15 of diabetesshowing accumulation of glomerular CD68+ macrophages. B: Treatment with GS-444217 reduced glomerular CD68+ macrophages indiabetic mice. C: Quantification of the number of glomerular CD68+ macrophages. D–G: Real-time PCR analysis of whole kidney mRNAlevels for CD68 (D), CCL2 (E), tumor necrosis factor (TNF)a (F), and KIM-1 (G). No Tx, no treatment; No STZ, controls; wk, week. *P < 0.05,**P < 0.01, ***P < 0.001.

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no detectable difference in fasting blood glucose orplasma insulin levels. The reason for this discrepancy isunclear. However, no change in HbA1c levels or otherparameters of diabetes was seen in the late-interventionstudy, demonstrating that halting established diabeticglomerulosclerosis with GS-444217 was not due to mod-ification of the diabetic state.

While there are several members of the MAP3K familythat can induce p38 activation, we have shown that ASK1plays a nonredundant and essential role in activating p38in the diabetic kidney. This is consistent with a role forASK1 in high glucose–induced p38 activation in culturedglomerular mesangial cells (36) and the various stressespresent in diabetes (e.g., oxidative stress, endoplasmicreticular stress, hyperglycemia, and angiotensin II) thatare known to activate ASK1/p38 signaling (37). However,activation of p38 MAPK signaling can operate indepen-dent of ASK1 in kidney cells as shown by the unalteredlipopolysaccharide and interleukin-1–induced p38 activa-tion and biological responses seen in Ask12/2 tubularepithelial cells (4). Thus, ASK1 is a context-dependent reg-ulator of p38 activation, which is particularly important insettings of oxidative stress owing to the regulation of ASK1activation by antioxidant proteins such as thioredoxin. Onelimitation of the current study is that currently availablecommercial antibodies are not sensitive enough to detectASK1 in tissue sections or for detection of ASK1 phosphor-ylation in kidney tissues.

ASK1/p38 signaling has been implicated in otheraspects of diabetes. ASK1/p38 signaling is involved instress-induced death of pancreatic b-cells and in the in-duction of endothelial cell senescence induced by highglucose (38,39). Ask12/2 mice have reduced levels of ma-ternal diabetes-induced endoplasmic reticulum stress inthe developing embryo (40). Indirect blockade of ASK1signaling can protect against diabetic cardiomyopathy(41,42), although transgenic mice overexpressing a kinasedead form of ASK1 are not protected from diabetic neu-ropathy (43). Therefore, pathological signaling by ASK1may be a common contributor to diabetic end organ dam-age in the pancreas, heart, and kidney.

In conclusion, treatment with an ASK1 inhibitor haltedthe progression of glomerulosclerosis and renal inflamma-tion and improved renal function in an aggressive model ofdiabetic nephropathy. This protective effect was attributedto blockade of ASK1-dependent p38 signaling. This studyidentifies ASK1 as a new therapeutic target in diabeticnephropathy, and a related ASK1 inhibitor is now beingtested in a phase 2 clinical trial (NCT02177786).

Funding. This study was partly funded by the National Health and MedicalResearch Council of Australia (APP1044289).Duality of Interest. This study was also funded by Gilead Sciences.G.H.T. was partly supported by Gilead Sciences. J.T.L. and D.G.B.are employees of Gilead Sciences. D.J.N.-P. acts as a consultant forGilead Sciences. Gilead Sciences is currently running a clinical trial

with an ASK1 inhibitor in patients with diabetic kidney disease(NCT02177786). No other potential conflicts of interest relevant to thisarticle were reported.Author Contributions. G.H.T., F.Y.M., and Y.H. performed the study.G.H.T., F.Y.M., Y.H., and D.J.N.-P. analyzed data. G.H.T. and D.J.N.-P. designedthe study. J.T.L. and D.G.B. provided the ASK1 inhibitor and pharmacokineticdata. G.H.T. and D.J.N.-P. wrote the manuscript, which was approved by allauthors. G.H.T. is the guarantor of this work and, as such, had full access to allthe data in the study and takes responsibility for the integrity of the data and theaccuracy of the data analysis.

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