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 abrogated 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. Immunostaining for fore and after a 6-week period on chow containing 0.1% phospho-p38, WT1, and collagen IV was performed on GS-444217. GS-444217 treatment was well tolerated by 4 mm paraffin-embedded sections, which were fixed in 4% diabetes.diabetesjournals.org Tesch and Associates 3905

2/2 formalin or methylcarn solution. Immunostaining for Since diabetes does not affect hypertension in Nos3 CD68 was performed on 5 mm paraformaldehyde-lysine- mice (25,26), we measured the effect of GS-444217 on 2/2 periodate–fixed cryostat sections. For antigen retrieval of blood pressure in nondiabetic Nos3 mice. Systolic 2/2 phospho-p38, dewaxed paraffin sections were heated in blood pressure in Nos3 mice was 133 6 5mmHg a microwave oven (800 watts for 12 min) in 10 mmol/L before treatment and was unchanged (134 6 5mmHg) sodium citrate buffer (pH 6.0) (22). For immunostaining, after 6 weeks of GS-444217 administration, demonstrat- sections were treated with 20% rabbit serum or 20% goat ing that GS-444217 does not affect hypertension in serum for 30 min and then incubated with primary anti- these mice. body in 3% BSA overnight at 4°C. Sections were then placed in 0.6% hydrogen peroxide in methanol for 20 Early Intervention With GS-444217 Inhibits the Development of Diabetic Renal Injury min to inactivate endogenous peroxidase. Bound primary 2/2 Groups of diabetic Nos3 mice were fed standard chow antibodies were detected using a standard ABC-peroxidase or chow with 0.1% GS-444217 from week 2 after STZ system: avidin-biotin block, biotinylated antibodies (rabbit injection until being killed on week 8. GS-444217 had anti-goat IgG, rabbit anti-rat IgG, or goat anti-mouse IgG), no effect upon fasting body weight, fasting blood glucose and ABC-peroxidase (Vector Laboratories, Burlingame, CA). levels, or plasma insulin levels, although a partial reduc- Sections were developed with 3,3-diaminobenzidine (Sigma) tion in HbA levels was evident in the drug-treated group to produce a brown color. CD68 sections were counter- 1c (Fig. 2). A substantial increase in urinary albumin excre- stained with hematoxylin to assist cell counting. Normal tion was evident on weeks 5 and 8 after STZ injection, goat serum or isotype-matched irrelevant IgG was used as which was not affected by GS-444217 treatment (Fig. 3A). negative control. There was also a reduction in the number of glomerular + Quantitation of Immunohistochemistry WT1 podocytes at week 8 after STZ, which was unaf- The number of CD68+ cells and WT1+ podocytes was fected by drug treatment (Fig. 3B). However, the decline counted in 30 hilar glomerular cross-sections (gcs) per in renal function at week 8 after STZ, as assessed by in- animal (3400). Glomerular collagen IV staining was quan- creased serum cystatin C levels, was significantly im- titated by computer image analysis (Image-Pro Plus, proved by drug treatment (Fig. 3C). 2/2 Media Cybernetics, Silver Spring, MD) in 50 hilar glomer- Nos3 mice with no treatment developed mild-to- ular cross-sections (3400) and expressed as the percent- moderate glomerulosclerosis by week 8 of diabetes, as age of glomerular area stained. All scoring was performed shown by increased PAS-stained glomerular deposits on blinded slides. and an increase in glomerular staining for collagen IV (Fig. 4). In addition, a significant glomerular infiltrate of Real-time PCR CD68+ macrophages was evident (Fig. 4). GS-444217 sig- Total RNA was extracted from whole kidney using Trizol nificantly reduced both glomerulosclerosis and glomerular (Invitrogen) and reverse transcribed with random primers macrophage infiltration (Fig. 4). using the Superscript First-Strand Synthesis kit (Invitrogen). Real-time PCR analysis was performed as previously Late Intervention With GS-444217 Improves Renal described (5,23). The Taqman probe and primer sequences Function and Halts Progressive Glomerulosclerosis in Established Diabetic Nephropathy are listed in Supplementary Table 1. 2/2 Groups of diabetic Nos3 mice were fed standard chow Statistical Analysis or chow with 0.1% GS-444217 from week 8 after STZ Statistical differences were analyzed by one-way ANOVA injection until being killed on week 15. In animals fed with Tukey multiple comparison posttest. Data were the standard diet, disease severity was maintained or in- recorded as mean 6 SEM with P , 0.05 considered sig- creased between weeks 8 and 15 (Figs. 5 and 6). GS- nificant. All analyses were performed using GraphPad 444217 had no effect upon fasting body weight, fasting Prism 6.0 (GraphPad Software, San Diego, CA). blood glucose levels, or HbA1c levels (Fig. 5). The diabetic mice had established albuminuria and raised serum RESULTS cystatin-C levels at week 8, when GS-444217 treatment Effect of ASK1 Inhibitor on p38 MAPK Signaling and commenced (Fig. 6). Drug treatment had no impact upon Blood Pressure established albuminuria or podocyte loss, but there was Consistent with previous studies (6,9,24), basal p38 phos- a significant reduction in the levels of serum cystatin-C, phorylation (activation) was detected in nondiabetic kid- suggesting that GS-444217 treatment improved renal ney by Western blot, while immunostaining identified function (Fig. 6). phosphorylated p38 in glomeruli, tubules, and the inter- There was a clear increase in diabetic glomeruloscle- stitium (Fig. 1A and E). A significant increase in p38 ac- rosis between weeks 8 and 15 in untreated diabetic 2/2 tivation in the kidney was evident at both weeks 8 and Nos3 mice as shown by glomerular collagen IV stain- 15 (Fig. 1). In contrast, treatment with GS-444217 over ing (Fig. 7A–C and E). This was associated with a sustained weeks 2–8 and over weeks 8–15 of diabetes caused a dra- increase in renal mRNA levels for profibrotic molecules matic inhibition of p38 activation in all cell types (Fig. 1). (collagens 1 and 4, fibronectin, transforming growth 3906 ASK1 in Diabetic Nephropathy Diabetes Volume 64, November 2015

Figure 1—Effect of ASK1 inhibitor (ASK1i) GS-444217 on p38 MAPK activation in the diabetic kidney. Western blot analysis of phosphorylated (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 from week 2 after STZ injection until being killed at week 8. Age-matched nondiabetic Nos32/2 mice were used as controls (No STZ). Western blot analysis (C) and quantification (D) of p-p38 in whole kidney samples in the late-intervention study. Groups of diabetic Nos32/2 mice were 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-matched nondiabetic Nos32/2 mice were used as controls (No STZ). Kidney immunostaining for phosphorylated p38 (brown) at week 8 (E–G) and week 15 (H–J) of diabetes. Immunostaining for phosphorylated p38 was seen in glomeruli, tubules, and the interstitium of nondiabetic Nos32/2 control kidneys at week 8 (E) and week 15 (H). An increase in p-p38 was evident in diabetic kidneys, particularly within glomeruli and dilated tubules at week 8 (F), and was similarly elevated at week 15 (I). GS-444217 treatment largely abrogated the phosphorylated p38 staining in diabetic kidneys at week 8 (G) and week 15 (J). Original magnification (E–G) 3250. ***P < 0.001. w, weeks. factor-b1, and plasminogen activator inhibitor-I) (Fig. 7F–J) necrosis factor-a)(Fig.8A–F).Inaddition,expressionof and a sustained glomerular CD68+ macrophage infiltrate the tubular damage marker kidney injury molecule-1 and increases in renal mRNA levels for proinflammatory (KIM-1) was elevated at week 8 of diabetes and remained molecules (chemokine CC motif ligand 2 [CCL2] and tumor elevated through to week 15 (Fig. 8G). diabetes.diabetesjournals.org Tesch and Associates 3907

significantly reduced p38 activation, albuminuria, podocyte loss, and glomerular collagen deposition and improved renal function (9). The current study significantly extends 2/2 these findings in two ways. First, diabetic Nos3 mice develop a much more severe form of glomerulosclerosis compared with the above two models (21), thereby providing a much sterner test of the ability of ASK1/p38 blockade to prevent diabetic glomerulosclerosis. Second, we performed an intervention study and demonstrated that GS-444217 could inhibit p38 activation, halt glomerulosclerosis, reduce glomerular inflammation, and improve renal 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 development of diabetes in Nos32/2 mice. Groups of diabetic Nos32/2 mice were fed standard chow (no treatment [No Tx]) or chow with 0.1% GS-444217 from week 2 after STZ injection until being killed on week 8. Age-matched nondiabetic Nos32/2 mice were used as controls (No STZ). Fasting body weight (A), fasting blood glucose (FBG) (B), HbA1c levels (C), and plasma insulin levels (D). NS, not signiﬁcant. ***P < 0.001.

GS-444217 treatment over weeks 8–15 prevented the increase in glomerular deposition of collagen IV and downregulated renal mRNA levels for all of the profibrotic molecules analyzed (Fig. 7D–J). In addition, GS-444217 treatment significantly reduced the glomerular macrophage infiltrate and reduced the renal mRNA expression of proinflammatory markers and tubular damage marker KIM-1 (Fig. 8).

DISCUSSION Administration of an ASK1 inhibitor, GS-444217, was shown to be effective in a mouse model of established diabetic nephropathy on the basis of halting progressive glomerulosclerosis, reducing inflammation, and improv- ing renal function despite ongoing hyperglycemia, hypertension, and albuminuria. The protective effect of ASK1 inhibitor treatment in diabetic nephropathy was attributed to the highly effective blockade of p38 activation—the major downstream target for ASK1 (14). GS-444217 is highly selective for ASK1, although as in all pharmacology studies, we cannot formally rule out unanticipated off-target effects. In- — creased p38 activation has been described in human and Figure 3 Effect of ASK1 inhibitor (ASK1i) GS-444217 on albuminuria, podocyte loss, and renal function in early diabetes. Groups – 2/2 experimental diabetic nephropathy (6 9), which was con- of diabetic Nos3 mice were fed standard chow (no treatment firmed in the current study. Administration of a p38 [No Tx]) or chow with 0.1% GS-444217 from week 2 after STZ in- 2/2 MAPK inhibitor has been shown to reduce albuminuria jection until being killed on week 8. Age-matched nondiabetic Nos3 fi mice were used as controls (No STZ). Urine albumin-to-creatinine ratio and prevent the development of mild glomerular brosis (ACR) (A), number of WT1+ podocytes per glomerular cross-section in nonhypertensive diabetic rats (10). In addition, dele- (B), and renal function measured by serum cystatin-C levels (C). NS, tion of the Mkk3 gene in db/db mice with type 2 diabetes not significant. **P < 0.05, ***P < 0.001. 3908 ASK1 in Diabetic Nephropathy Diabetes Volume 64, November 2015

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 mice with no treatment. C: Treatment with GS-444217 over weeks 2–8 reduced glomerular PAS-stained deposits. D–F: Immunostaining for collagen 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 suppressed glomerular 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 in untreated mice after 8 weeks of diabetes. I: Treatment with GS-44217 reduced the number of glomerular CD68+ cells in diabetic mice. J:Image analysis of the area of glomerular collagen IV staining (glom area). K: Quantification of the number of glomerular CD68+ macrophages. Original magnification (A–I) 3400. *P < 0.05, **P < 0.01, ***P < 0.001. trial evaluating an ASK1 inhibitor, GS-4997, in patients A second mechanism whereby ASK1 inhibition reduced with stage 3/4 diabetic kidney disease (27). diabetic glomerular damage is through the inhibition of The ability of GS-444217 to reduce collagen deposition macrophage infiltration and inhibition of mRNA levels of in the diabetic glomerulus is consistent with previous proinflammatory mediators. Previous studies have shown studies in which p38 inhibitors have been shown to that macrophages cause glomerular injury in experiment reduce renal fibrosis in nondiabetic models of kidney models of diabetic nephropathy (32,33). This is consistent 2/2 disease (19,28,29). In addition, Ask1 mice show a pro- with the reduction in CCL2 mRNA levels and macrophage found suppression of p38 activation and tissue fibrosis in infiltration seen in the tubulointerstitium in the obstructed 2/2 models of both renal and cardiac disease (4,30,31). kidney in Ask1 mice (4). diabetes.diabetesjournals.org Tesch and Associates 3909

Figure 5—Effect of ASK1 inhibitor (ASK1i) GS-444217 on the progression of diabetes in Nos32/2 mice. Groups of diabetic Nos32/2 mice were fed standard chow (no treatment [No Tx]) or chow with 0.1% GS-444217 from week 8 after STZ injection until being killed 2/2 on week 15. Age-matched nondiabetic Nos3 mice were used as Figure 6—Effect of ASK1 inhibitor (ASK1i) GS-444217 on albumin- controls (No STZ). Fasting body weight (FBW) (A), fasting blood uria, podocyte loss, and renal function in progressive diabetes. 2/2 glucose (FBG) (B), and HbA1c levels (C). Groups of diabetic Nos3 mice were fed standard chow (no treatment [No Tx]) or chow with 0.1% GS-444217 from week 8 after STZ injection until being killed on week 15. Age-matched nondiabetic Nos32/2 mice were used as controls (No STZ). Urine albumin-to- The aggressive nature of disease development in A + Nos32/2 creatinine ratio (ACR) ( ), number of WT1 podocytes per glomerular diabetic mice is attributed to the presence of cross-section (B), and renal function measured by serum cystatin-C hypertension. However, the ASK1 inhibitor protected levels (C). NS, not significant; wk, week. ***P < 0.001 vs. STZ-No Tx. against diabetic glomerulosclerosis despite having no ef- 2/2 fect on hypertension in Nos3 mice. This is consistent 2/2 with previous studies on Ask1 mice, which are protected from end organ damage in the setting of hyperten- The loss of podocytes and development of albuminuria 2/2 sion. For example, in models of angiotensin II and in diabetic Nos3 mice were unaffected by ASK1 inhib- 2/2 mineralocorticoid-driven hypertension, Ask1 mice itor treatment in our early- and late-intervention studies. had reduced cardiac fibrosis despite unaltered hyperten- This suggests that activation of ASK1/p38 signaling is not sion (30,31). In the current study, ASK1 inhibition with involved in the mechanism of diabetic albuminuria. How- GS-444217 appears to reduce renal injury and improve ever, there are some limitations in this interpretation. 2/2 renal function by directly decreasing inflammation and Recent studies of Nos3 mice has shown that they fibrosis in the diabetic kidney. are highly susceptible to glucose-induced podocyte damage 3910 ASK1 in Diabetic Nephropathy Diabetes Volume 64, November 2015

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 8–15 prevents 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; No STZ, controls. *P < 0.05, **P < 0.01, ***P < 0.001. diabetes.diabetesjournals.org Tesch and Associates 3911

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:ANos32/2 kidney at week 15 of diabetes showing accumulation of glomerular CD68+ macrophages. B: Treatment with GS-444217 reduced glomerular CD68+ macrophages in diabetic mice. C: Quantiﬁcation of the number of glomerular CD68+ macrophages. D–G: Real-time PCR analysis of whole kidney mRNA levels 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.

and show marked albuminuria 2 weeks after STZ admin- studies in both diabetic and nondiabetic kidney disease istration (34), which was the time at which we began have shown that p38 inhibitors can reduce albuminuria ASK1 inhibitor treatment in the early-intervention study. (9,10,28,35), arguing that ASK1/p38 signaling may con- Therefore, it is possible that ASK1 inhibition may have tribute to albuminuria in some contexts. been commenced too late to have an effect on the induc- Early intervention with the ASK1 inhibitor saw a re- 2/2 tion of podocyte damage/loss and albuminuria. Previous duction in HbA1c levels in diabetic Nos3 mice despite 3912 ASK1 in Diabetic Nephropathy Diabetes Volume 64, November 2015 no detectable difference in fasting blood glucose or with an ASK1 inhibitor in patients with diabetic kidney disease plasma insulin levels. The reason for this discrepancy is (NCT02177786). No other potential conflicts of interest relevant to this article were reported. unclear. However, no change in HbA1c levels or other parameters of diabetes was seen in the late-intervention 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. designed study, demonstrating that halting established diabetic the study. J.T.L. and D.G.B. provided the ASK1 inhibitor and pharmacokinetic glomerulosclerosis with GS-444217 was not due to mod- fi data. G.H.T. and D.J.N.-P. wrote the manuscript, which was approved by all i cation of the diabetic state. authors. G.H.T. is the guarantor of this work and, as such, had full access to all While there are several members of the MAP3K family the data in the study and takes responsibility for the integrity of the data and the that can induce p38 activation, we have shown that ASK1 accuracy of the data analysis. plays a nonredundant and essential role in activating p38 in the diabetic kidney. This is consistent with a role for References ASK1 in high glucose–induced p38 activation in cultured 1. Lv J, Perkovic V, Foote CV, Craig ME, Craig JC, Strippoli GF. Antihyper- glomerular mesangial cells (36) and the various stresses tensive agents for preventing diabetic kidney disease. Cochrane Database Syst present in diabetes (e.g., oxidative stress, endoplasmic Rev 2012;12:CD004136 reticular stress, hyperglycemia, and angiotensin II) that 2. Chang PC, Chen TH, Chang CJ, Hou CC, Chan P, Lee HM. Advanced gly- are known to activate ASK1/p38 signaling (37). However, cosylation end products induce inducible nitric oxide synthase (iNOS) expression – activation of p38 MAPK signaling can operate indepen- via a p38 MAPK-dependent pathway. Kidney Int 2004;65:1664 1675 3. Kang MJ, Wu X, Ly H, Thai K, Scholey JW. Effect of glucose on stress- dent of ASK1 in kidney cells as shown by the unaltered – activated protein kinase activity in mesangial cells and diabetic glomeruli. Kidney lipopolysaccharide and interleukin-1 induced p38 activa- Int 1999;55:2203–2214 Ask12/2 tion and biological responses seen in tubular 4. Ma FY, Tesch GH, Nikolic-Paterson DJ. ASK1/p38 signaling in renal tubular epithelial cells (4). Thus, ASK1 is a context-dependent reg- epithelial cells promotes renal fibrosis in the mouse obstructed kidney. Am J ulator of p38 activation, which is particularly important in Physiol Renal Physiol 2014;307:F1263–F1273 settings of oxidative stress owing to the regulation of ASK1 5. Ma FY, Tesch GH, Ozols E, Xie M, Schneider MD, Nikolic-Paterson DJ. TGF- activation by antioxidant proteins such as thioredoxin. One b1-activated kinase-1 regulates inflammation and fibrosis in the obstructed limitation of the current study is that currently available kidney. Am J Physiol Renal Physiol 2011;300:F1410–F1421 commercial antibodies are not sensitive enough to detect 6. Adhikary L, Chow F, Nikolic-Paterson DJ, et al. Abnormal p38 mitogen- ASK1 in tissue sections or for detection of ASK1 phosphor- activated protein kinase signalling in human and experimental diabetic ne- – ylation in kidney tissues. phropathy. Diabetologia 2004;47:1210 1222 7. Komers R, Lindsley JN, Oyama TT, Cohen DM, Anderson S. Renal p38 MAP ASK1/p38 signaling has been implicated in other kinase activity in experimental diabetes. Lab Invest 2007;87:548–558 aspects of diabetes. ASK1/p38 signaling is involved in 8. Verzola D, Gandolfo MT, Ferrario F, et al. Apoptosis in the kidneys of pa- stress-induced death of pancreatic b-cells and in the in- tients with type II diabetic nephropathy. Kidney Int 2007;72:1262–1272 duction of endothelial cell senescence induced by high 9. Lim AK, Nikolic-Paterson DJ, Ma FY, et al. Role of MKK3-p38 MAPK sig- 2/2 glucose (38,39). Ask1 mice have reduced levels of ma- nalling in the development of type 2 diabetes and renal injury in obese db/db ternal diabetes-induced endoplasmic reticulum stress in mice. Diabetologia 2009;52:347–358 the developing embryo (40). Indirect blockade of ASK1 10. Jung DS, Li JJ, Kwak SJ, et al. FR167653 inhibits fibronectin expression signaling can protect against diabetic cardiomyopathy and apoptosis in diabetic glomeruli and in high-glucose-stimulated mesangial – (41,42), although transgenic mice overexpressing a kinase cells. Am J Physiol Renal Physiol 2008;295:F595 F604 “ ” dead form of ASK1 are not protected from diabetic neu- 11. Hammaker D, Firestein GS. Go upstream, young man : lessons learned from the p38 saga. Ann Rheum Dis 2010;69(Suppl. 1):i77–i82 ropathy (43). Therefore, pathological signaling by ASK1 12. Keshet Y, Seger R. The MAP kinase signaling cascades: a system of may be a common contributor to diabetic end organ dam- hundreds of components regulates a diverse array of physiological functions. age in the pancreas, heart, and kidney. Methods Mol Biol 2010;661:3–38 In conclusion, treatment with an ASK1 inhibitor halted 13. Shiizaki S, Naguro I, Ichijo H. Activation mechanisms of ASK1 in response to the progression of glomerulosclerosis and renal inflamma- various stresses and its significance in intracellular signaling. Adv Biol Regul tion and improved renal function in an aggressive model of 2013;53:135–144 diabetic nephropathy. This protective effect was attributed 14. Ichijo H, Nishida E, Irie K, et al. Induction of apoptosis by ASK1, a mam- to blockade of ASK1-dependent p38 signaling. This study malian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science identifies ASK1 as a new therapeutic target in diabetic 1997;275:90–94 nephropathy, and a related ASK1 inhibitor is now being 15. Tobiume K, Inage T, Takeda K, Enomoto S, Miyazono K, Ichijo H. Molecular cloning and characterization of the mouse apoptosis signal-regulating kinase 1. tested in a phase 2 clinical trial (NCT02177786). Biochem Biophys Res Commun 1997;239:905–910 16. Gerits N, Kostenko S, Moens U. In vivo functions of mitogen-activated protein kinases: conclusions from knock-in and knock-out mice. Transgenic Res Funding. This study was partly funded by the National Health and Medical 2007;16:281–314 Research Council of Australia (APP1044289). 17. Tobiume K, Matsuzawa A, Takahashi T, et al. ASK1 is required for sustained Duality of Interest. This study was also funded by Gilead Sciences. activations of JNK/p38 MAP kinases and apoptosis. EMBO Rep 2001;2:222–228 G.H.T. was partly supported by Gilead Sciences. J.T.L. and D.G.B. 18. Furuichi K, Wada T, Iwata Y, et al. Administration of FR167653, a new anti- are employees of Gilead Sciences. D.J.N.-P. acts as a consultant for inflammatory compound, prevents renal ischaemia/reperfusion injury in mice. Gilead Sciences. Gilead Sciences is currently running a clinical trial Nephrol Dial Transplant 2002;17:399–407 diabetes.diabetesjournals.org Tesch and Associates 3913

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