Lysophosphatidic Acid Receptor Antagonism Protects Against Diabetic Nephropathy in a Type 2 Diabetic Model

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Lysophosphatidic Acid Receptor Antagonism Protects against Diabetic Nephropathy in a Type 2 Diabetic Model

† ‡ †‡ Ming-Zhi Zhang,* Xin Wang,* Haichun Yang, Agnes B. Fogo, Brian J. Murphy,§ †| Robert Kaltenbach,§ Peter Cheng,§ Bradley Zinker,§ and Raymond C. Harris*

*Division of Nephrology and Hypertension, Department of Medicine, †Vanderbilt Center for Kidney Disease, and ‡Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee; §Fibrosis Discovery Biology or Chemistry, Bristol-Myers Squibb, Pennington, New Jersey; and |United States Department of Veterans Affairs, Nashville, Tennessee

ABSTRACT Lysophosphatidic acid (LPA) functions through activation of LPA receptors (LPARs). LPA–LPAR signaling has been implicated in development of fibrosis. However, the role of LPA–LPAR signaling in development of diabetic nephropathy (DN) has not been studied. We examined whether BMS002, a novel dual LPAR1 and LPAR3 antagonist, affects development of DN in endothelial nitric oxide synthase-knockout db/db mice. Treatment of these mice with BMS002 from 8 to 20 weeks of age led to a significant reduction in albuminuria, similar to that observed with renin-angiotensin system inhibition (losartan plus enalapril). LPAR inhibition also prevented the decline in GFR observed in vehicle-treated mice, such that GFR at week 20 differed significantly between vehicle and LPAR inhibitor groups (P,0.05). LPAR inhibition also reduced histologic glomerular injury; decreased the expression of profibrotic and fibrotic components, including fibronectin, a-smooth muscle actin, connective tissue growth factor, collagen I, and TGF-b;and reduced renal macrophage infiltration and oxidative stress. Notably, LPAR inhibition slowed podocyte loss (podocytes per glomerulus 6SEM at 8 weeks: 667640, n=4; at 20 weeks: 364618 with vehicle, n=7, and 536612 with LPAR inhibition, n=7; P,0.001 versus vehicle). Finally, LPAR inhibition minimized the production of 4-hydroxynonenal (4-HNE), a marker of oxidative stress, in podocytes and increased the phosphorylation of AKT2, an indicator of AKT2 activity, in kidneys. Thus, the LPAR antagonist BMS002 protects against GFR decline and attenuates development of DN through multiple mechanisms. LPAR antagonism might provide complementary beneficial effects to renin-angiotensin system inhibition to slow progression of DN.

J Am Soc Nephrol 28: 3300–3311, 2017. doi: https://doi.org/10.1681/ASN.2017010107

Lysophosphatidic acid (LPA) is a small and struc- receptors, LPA1–6 or LPAR1–6. There are at least two turally simple glycerophospholipid, with one fatty pathways for LPA production: a direct action of the acid and a phosphate group as its polar head. It is a extracellular enzyme autotaxin (ectonucleotide normal serum constituent and binds to albumin with high afﬁnity. Although originally considered Received January 31, 2017. Accepted June 19, 2017. as a component of the plasma membrane and an Published online ahead of print. Publication date available at intermediate in lipid biosynthesis, there is increas- www.jasn.org. ing evidence that LPA can mediate a wide range Correspondence: Dr. Raymond C. Harris, Department of Medi- of cellular processes, including cell migration, cine, Vanderbilt University School of Medicine, C-3321 Medical Center North, Nashville, TN 37232, or Dr. Ming-Zhi Zhang, De- proliferation and cytoprotection, cytokine and partment of Medicine, Vanderbilt University School of Medicine, chemokine production, smooth muscle contrac- S3223 Medical Center North, Nashville, TN 37232. E-mail: ray. tion, and platelet aggregation.1 LPA acts through [email protected] or [email protected] activation of a family of speciﬁc G protein–coupled Copyright © 2017 by the American Society of Nephrology

3300 ISSN : 1046-6673/2811-3300 JAmSocNephrol28: 3300–3311, 2017 www.jasn.org BASIC RESEARCH pyrophosphatase/phosphodiesterase family member 2, ENPP2) development of diabetic nephropathy in association with po- on plasma lysophosphatidylcholine to produce LPAand choline, docyte protection and increased expression of reparative or intracellularly either by sequential activation of phospho- M2 macrophages. lipase D and phosphatidic acid–specific phospholipase A or by acylglycerol kinase. Essentially all extracellular LPA results from autotaxin activity.2 RESULTS LPA has been reported to stimulate proliferation and inhibit apoptosis in mouse renal proximal tubular cells,3 We initially determined the effect of diabetes on the expression fibroblasts,4 and mesangial cells,5,6 In cultured proximal of autotaxin and LPARs in the kidney. Immunoblotting showed tubular cells, LPA stimulates the secretion of profibrotic that the expression levels of LPAR1, LPAR3, and autotaxin, the cytokines CTGF and PDGF-B.7 LPA production increases major source of LPA released from cells, were markedly in- at sites of injury and inflammation and can serve as a potent creased in kidneys of an accelerated model of type 2 diabetic 2 2 proinflammatory and profibrotic mediator. In mouse kid- nephropathy (eNOS / db/db mice) compared with sex- and neys, the primary LPARs are LPA1, LPA2, and LPA3.8,9 LPA age-matched nondiabetic controls (Figure 1A). In nondiabetic has been reported to contribute to the development of renal mice, LPAR1 and LPA3 were primarily expressed in tubular tubulointerstitial fibrosis in a unilateral ureteral obstruc- epithelial cells, with minimal expression in the glomerulus. tion model, as well as in the development of skin and lung However, both LPAR1 and LPAR3 expression increased in 2 2 fibrosis. Either pharmacologic or genetic inhibition of LPA glomeruli of eNOS / db/db mice. Double immunofluores- receptor 1 (LPAR1) attenuates development of fibrosis in cence staining with nephrin (a marker of podocytes) indicated these models.8,10 However, the role of the autotoxin/LPA/ that the increased expression of LPAR1 and LPAR3 in glomer- LPA receptor (LPAR) system in diabetic nephropathy has uli was primarily in podocytes but was also present in other not been previously studied. This study investigated the glomerular cell types (Figure 1B). Similarly in nondiabetic effect of an LPAR antagonist in a model of accelerat- mice, autotaxin was expressed at low levels in podocytes, and 2 2 ed type 2 diabetic nephropathy, endothelial nitric oxide expression levels increased markedly in eNOS / db/db 2 2 synthase-knockout (eNOS / ) db/db mice. We found mice (Figure 1C). Furthermore, kidney expression of LPA3 2 2 that LPAR antagonism was effective in attenuating and autotaxin increased from 8 to 24 weeks of age in eNOS /

2 2 Figure 1. Podocyte LPA signaling is activated in eNOS / db/db mice. (A) The renal protein levels of the LPAR1, LPAR3, and autotaxin, 2 2 the main extracellular enzyme for LPA biosynthesis, were signiﬁcantly higher in eNOS / db/db mice than in control BKS mice. n=4 in 2 2 BKS group and n=5 in eNOS / db/db group. (B–D) LPAR1 (B), LPAR3 (C), and autotaxin (D) were localized to podocytes of mouse 2 2 glomerulus and their expression was all higher in eNOS / db/db mice than in age-matched wild-type mice (20 weeks old). Original magniﬁcation, 3400 for all. ***P,0.001.

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Figure 1. Continued. db/db mice (Supplemental Figure 1). Therefore, the LPA/LPAR catheterization measurement in conscious mice (mean BP: axis is activated in kidneys (particularly in podocytes) in 139.662.6 versus 136.561.1 mm Hg of vehicle; P=0.34, n=3 2 2 eNOS / db/db mice. in each group). To determine the potential role of LPAR activation in the mediation of diabetic kidney disease, we administered a rela- tively specific LPAR1/LPAR3 antagonist, BMS002 (a lysophos- 2 2 phatidic acid receptor inhibitor [LPARI]), to eNOS / db/db mice from 8 to 20 weeks of age. As a comparator, we treated a subset of mice with a combination of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker (ACEI/ ARB) (10 mg/kg per day losartan and 10 mg/kg per day enalapril,anRASinhibitor).Bodyweightswerecomparable among the three groups at both 8 and 20 weeks of age (Sup- plemental Figure 2). RAS inhibition led to a significant decrease of albuminuria by 6 weeks of treatment, which was sustained without further decrease over the subsequent 6 weeks of treatment (Figure 2). In contrast, LPARI induced only a numerical but nonsignificant decrease in albuminuria Figure 2. Effect of LPAR antagonist on albuminuria. Treatment after 6 weeks of treatment but induced a further, significant with losartan plus enalapril (RASI) led to rapid and marked decreases in albuminuria, whereas treatment with a LPARI for decrease in albuminuria by the end of 12 weeks of treatment both LPAR1 and LPAR3 caused a delayed but marked reduction of (Figure 2). Within 2 weeks of treatment, the RAS inhibitors albuminuria similar to that seen in RAS inhibition group, with fi signi cantly decreased systolic BP (SBP), whereas BMS002 minimal effect on hyperglycemia. *P,0.05; **P,0.01; ***P,0.001 had no significant early effect on BP as measured with a tail- versus baseline; ##P,0.01; ###P,0.001 versus corresponding vehi- cuff microphonic manometer (Supplemental Figure 3). The cle group; n=10 in each group. Bonferroni t tests were used for effect of BMS002 on BP was further validated using carotid statistical analysis.

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In vehicle-treated mice, GFR, measured by FITC inulin, As both LPAR and RAS inhibition attenuated glomerulo- 2 2 decreased significantly from 8 to 20 weeks (0.2260.01 versus sclerosis in eNOS / db/db mice, we investigated whether 0.1660.02 ml/min per mouse, n=10; P,0.02). In contrast, podocyte number was affected. qPCR data indicated that the there was no decrease in GFR in the LPARI group (0.246 mRNA levels of podocin and nephrin, two specific markers of 0.02 versus 0.2660.02 ml/min per mouse, n=10; NS) or in podocytes, were markedly higher in LPAR inhibitor–treated the RAS inhibitor group (0.2360.03 versus 0.2460.03 ml/min kidneys than in either vehicle- or RAS inhibitor–treated kid- 2 2 per mouse, n=10; NS). Therefore, after treatment for 12 weeks, neys (Figure 7A). Compared with 8-week-old eNOS / db/db GFR was significantly higher in both LPARI and RAS inhibitor mice, vehicle-treated mice had lost approximately 50% of groups than the vehicle-treated group (Figure 3A). their podocytes by 20 weeks of age, indicated by WT-1 stain- At 20 weeks, there was significantly decreased glomerulo- ing (from 667640 to 364618 podocytes per glomerulus; n=4 sclerosis in both LPARI treated- and RAS inhibitor–treated in 8-week-old group and n=7 in 20-week-old vehicle-treated mice compared with vehicle-treated mice (Figure 3B). LPARI group; P,0.001) (Figure 7B). In contrast, LPAR inhibitor– also decreased the amount of total fibrosis (glomeruli treated mice had significantly less podocyte loss (536612 and tubulointerstitium) compared with vehicle (2.176 podocytes per glomerulus, n=7; P,0.001 versus 20-week- 0.17% versus 1.0260.10%, n=10; P,0.001) (Figure 4A). In- old vehicle-treated group). Similar to kidney nephrin and terestingly, LPARI was also more effective than RAS inhibition podocin mRNA data, RAS inhibition did not prevent the de- at attenuating total fibrosis (1.4760.12, n=10; P,0.01 versus crease in podocyte number seen in this model of diabetic BMS002 group). LPARI treatment decreased mRNA expres- kidney injury (337618 podocytes per glomerulus, n=6; NS ver- sion of fibronectin, collagen I, TGF-b, CTGF and a-smooth sus vehicle-treated mice; P,0.001 versus LPARI group). Our muscle actin (a-SMA) (Figure 4B), and there were similar previous study had shown that the angiotensin-converting decreases in expression of immunoreactive fibronectin, col- enzyme inhibitor (ACEI) captopril and a triple therapy of hy- lagen, and a-SMA, further indicating LPARI-mediated inhi- dralazine, reserpine, and hydrochlorothiazide decreased BP in 2 2 bition of profibrotic pathways (Figure 4B). eNOS / db/db mice to similar levels.11 On further analysis Inflammation contributes to development of diabetic ne- of these results, we found that the podocyte number per glomer- phropathy. Both LPAR antagonism and RAS inhibition led to ular section in both treated groups was similar to that of un- marked reduction in renal macrophage infiltration, as indi- treated mice, indicating that either lowering BP or inhibition of cated by decreased protein and mRNA levels of F4/80, a marker angiotensin II production with ACEI did not prevent loss of of macrophages (Figure 5, A and B). Although macrophage podocytes (Supplemental Figure 5). infiltration decreased, renal mRNA levels of IL-4Ra and 4-HNE is a marker of oxidative stress and represents the 15-lipoxygenase, two markers of anti-inflammatory and re- major products of lipid peroxidation. 4-HNE immunostaining parative M2 phenotypic macrophages, were significantly was apparent in podocytes and other cell types of glomeruli in 2 2 higher in both LPAR inhibitor–treated mice and RAS inhibitor– 20-week-old vehicle-treated eNOS / db/db mice, but was treated mice (Figure 5C). Both LPAR and RAS inhibition also markedly decreased in LPAR inhibitor–treated mice (Figure suppressed renal T cell infiltration (Figure 6A); decreased re- 8A). Recently AKT2 activation has been reported to play nal mRNA levels of Th1/M1 cytokines/chemokines, including an essential role in podocyte protection after chronic kidney IL-1a,IL-b,IFN-g, CCL-2, IL-23, and IL-6 (Figure 6B); and injury.12 We determined that renal AKT2 activity was signif- 2 2 reduced oxidative stress, as indicated by nitrotyrosine icantly lower in eNOS / db/db mice than age- and sex- immunostaining (Supplemental Figure 4). matched BKS controls (Figure 8B), and LPAR inhibition led

Figure 3. Antagonism of LPARs preserved GFR. (A) From 8 to 20 weeks of age, GFR markedly decreased in vehicle group, but was stable in RAS inhibition (RASI) and LPARI groups, with higher GFR in RASI and LPARI groups than in the vehicle group at 20 weeks of age. *P,0.05 versus corresponding baseline; #P,0.05 versus vehicle group at 20 weeks of age; n=10 in each group. (B) Both RASI and LPARI attenuated glomerular injury. n=8–10 in each group. t test and Bonferroni t tests were used for statistical analysis. **P,0.01; ***P,0.001 versus vehicle group.

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Figure 4. Antagonism of LPARs attenuated the development of diabetic nephropathy. (A) LPARIs and renin-angiotensin system inhibitors (RASIs) markedly attenuated the development of glomerular and tubulointerstitial fibrosis, as indicated by Picrosirius red staining and Masson trichrome staining and quantification. ***P,0.001 versus vehicle group; #P,0.05 versus RASI group; n=10ineachgroup. (B) Quantitative PCR and immunostaining showed that both RAS inhibition and LPAR antagonism significantly inhibited renal expression levels of profibrotic and fibrotic components, including collagen I, a-SMA (marker of myofibroblasts), connective tissue growth factor (CTGF), fibronectin (FN), and TGF-b. Original magnification, 3160 for Masson trichrome. Original magnification, 3400 for all others. Bonferroni t tests were used for statistical analysis. *P,0.05; **P,0.01; ***P,0.001 versus vehicle group; #P,0.05; ##P,0.01 versus RASI group; n=6 in each group.

2 2 to marked increases in renal AKT2 activity in eNOS / db/db we confirmed that both autotaxin and LPAR3 were expressed in mice (Figure 8C). GSK3 is a critical downstream element of podocytes, as well as other cell types of glomerulus in diabetic the AKT cell survival pathway and AKT2 deficiency led human kidneys. However, LPAR1 was primarily expressed in to attenuated GSK3a activation in a CKD model of renal tubular epithelial cells, with minimal expression in glomerulus ablation.12 We found that p-GSK3a (activated GSK3a)expres- in diabetic human kidneys (Figure 9). sion increased in podocytes of mice with LPAR inhibition (Figure 8D). To validate whether LPA signaling is activated in human DISCUSSION diabetic kidney, we performed immunohistochemistry to determine expression in both normal and diabetic human The major findings in this study include the following: (1) kidneys. We determined that expression of autotaxin, podocytes have an intact autotaxin/LPA/LPAR system, which 2 2 LPAR1, and LPAR3 was minimal in normal human kidneys, is activated in eNOS / db/db mice; (2) similar to inhibition but their expression all increased in diabetic human kidneys, of RAS, inhibition of LPAR1 and LPAR3 preserved GFR and particularly in epithelia (Supplemental Figure 6). Using double attenuated development of diabetic nephropathy, as indicated immunofluorescence with nephrin as a marker of podocytes, by decreases in albuminuria, fibrosis, expression levels of

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inflammation.20 Although total renal macrophage number was markedly reduced in mice treated with the LPAR antagonist or with ACEI and ARB, the expression level of anti-inflammatory M2 markers increased whereas that of proinflammatory M1 markers decreased. Therefore, antagonism of LPAR or RAS not only inhibited renal macrophage infiltration but also polarized macrophage to an M2 phenotype, leading to protection against diabetic nephropathy. Diabetes causes microvascular dysfunction, which contributes to development of diabetic nephropathy. Alterations in the expression and/or activity of eNOS are associated with development and/or progression of diabetic nephropathy,21 and eNOS deficiency induces acceleration of development of diabetic nephropathy.11,15,22,23 2 2 eNOS / db/db mice develop an accelerat- Figure 5. Antagonism of LPARs affected renal macrophage infiltration and polariza- ed diabetic nephropathy with distinct fea- tion. (A and B) Both RAS inhibition and LPAR antagonism suppressed renal macrophage infiltration, as indicated by reduction in F4/80 staining (marker of macrophages) tures of progressive nephropathy similar to (A) and mRNA levels (B). Original magnification, 3400. ***P,0.001 versus vehicle human diabetic nephropathy, including fi group, n=6 in each group. (C) Renal mRNA levels of IL-4Ra and 15-lipoxygenase early and signi cant albuminuria, mesan- (15-LOX), two M2 phenotypic markers of macrophages, were markedly higher in both giolysis, arteriolar hyalinosis, FSGS, and LPAR I and RASI groups than the vehicle group. Bonferroni t tests were used for nodular glomerulosclerosis, as well as statistical analysis. **P,0.01; ***P,0.001 versus vehicle group; #P,0.05; ##P,0.01 moderate hypertension.22 Our previous versus RASI group; n=6 in each group. study indicated that either BP control with triple therapy (hydralazine, reserpine, hydrochlorothiazide) or angiotensin- fibrotic and profibrotic components (collagen I, a-SMA, converting enzyme inhibition with captopril attenuated pro- CTGF, fibronectin and TGF-b), and decreased immune cell gression of diabetic nephropathy in this model.11 In contrast, infiltration; (3) inhibition of LPARs slowed podocyte loss in we did not detect any effect of the LPAR inhibitor on the 2 2 2 2 eNOS / db/db mice, and preservation of AKT2 activity may hypertension seen in the eNOS / db/db mice. contribute to this protective effect; and (4) LPA signaling is ac- As noted above, LPAR antagonism led to partial preserva- tivated in human diabetic kidneys, particularly in podocytes. tion of podocyte number. Unexpectedly, in this study, we failed Serum autotaxin levels are associated with proteinuria and to observe prevention of podocyte loss with RAS inhibition, kidney lesions in type 2 diabetic patients with biopsy-proven unlike previous reports in other experimental models of di- diabetic nephropathy,13 but the increase in expression of both abetic nephropathy, as well as other models of experimental autotaxin and LPARs in glomerular cells in the diabetic kidney, glomerular disease.24–26 Of note, these models did not have especially podocytes, suggests a potential role for autocrine eNOS deficiency. eNOS activity has been reported to play and/or paracrine LPAR activation as a mediator of injury. an important role to maintain podocyte integrity. eNOS LPA can also transactivate the EGF receptor,14 and inhibition of deficiency causes podocyte cellular hypertrophy, lysosomal EGF receptor activity protects against diabetic nephropathy.15–17 enlargement, vacuolization, microvillus formation, increased Diabetic nephropathy is known to be a proinflammatory mitochondrial fragmentation, and reduction of ATP produc- 2 2 state, and immune cell infiltration, particularly macrophages, tion.27 In eNOS / db/db mice, ultrastructural evidence of plays an important in the development and progression of podocyte injury is evident as early as 8 weeks of age, with diabetic nephropathy. Renal macrophages from mice with accumulation of vacuoles, pseudocysts, foot process efface- STZdiabeteshavegreaterexpressionofproinflammatory ment, and electron-dense droplets within the cytoplasm.21 M1 markers than anti-inflammatory M2 markers.18 In this Angiotensin II causes oxidative stress due to eNOS uncou- study, inhibition of either LPARs or RAS markedly decreased pling in both endothelial cells and mesangial cells, leading to renal infiltration of macrophages and lymphocytes (Figures 5 subsequent kidney injury. The protection of RAS inhibition and 6). Of note, LPA is a noncytokine survival factor for against diabetic nephropathy is due, in part, to restoration of macrophages.19 The autotaxin/LPA axis has also been re- eNOS coupling and subsequent decreased oxidative ported to be a novel regulator of lymphocyte homing and stress.28–30

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Inhibition of LPARs appears to protect 2 2 against diabetic nephropathy in eNOS / db/db mice, at least in part through mechanisms different than those that mediate protection with RAS inhibition. RAS inhibition led to a quick initial reduction in albuminuria, whereas LPAR inhibition led to a gradual reduction in albuminuria. Our previous study addressed the role of eNOS deficiency and endothelial dysfunction in initiation of podocyte injury in this model of diabetic nephropathy.21 We propose that RAS inhibition may decrease albuminuria 2 2 in eNOS / db/db mice because of alterations in SBP and glomerular hemodynam- ics. The slower decrease in proteinuria and the lack of BP alterations with LPAR inhibition suggest that hemodynamic alterations are not paramount and the protective effect may be due predominantly to decreased inflammation and fibrosis. Furthermore, the ability of the LPAR inhib- 2 2 itor to protect podocytes in the eNOS / db/db mice suggests a direct cytoprotective protective effect on podocytes rather than an indirect effect caused by decreased endothelial dysfunction. In this regard, AKT2 has been reported to be expressed primarily in podocytes in kidney and to play a key role in protection against podocyte loss during chronic kidney injury.12 We found that 2 2 AKT2 activity was reduced in eNOS / db/db mice, and LPAR inhibitor preserved AKT2 activity and its downstream p- GSK3a activity. Recently, Li et al.31 reported that renal cortical mRNA and protein levels of autotaxin and LPAR1, but not LPAR2 and LPAR3, increased significantly in db/db mice compared with wild-type mice. In- creases in autotaxin and LPAR1 levels in db/db mice or in mesangial cells cultured in high glucose media were inhibited by ki16425, a dual LPAR1 and LPAR3 antagonist. They further found that ki16425 attenuated the development of diabetic nephropathy in db/db mice, possibly due to Figure 6. Antagonism of LPARs attenuated renal levels of proinflammatory cytokines/ inhibiting the LPAR1 signaling–mediated chemokines. (A) Both RAS inhibition and LPAR antagonism markedly suppressed renal TGF-b via GSK3b phosphorylation and Tlymphocyteinfiltration, as indicated by reduction of CD3 staining and quantification SREBP1 activation in mesangial cells. In 2 2 as well as mRNA levels. Original magnification, 3250. **P,0.01; ***P,0.001 versus our study, we used eNOS / db/db mice, vehicle group, n=6 in each group. (B) RAS inhibition and LPAR antagonism markedly an accelerated type 2 diabetic model. In fl reduced renal mRNA levels of proin ammatory cytokines/chemokines, including these mice, the protein levels of autotaxin, IL-1a,IL-1b, IL-23, IL-6, INF-g, and CCL2. Bonferroni t tests were used for statistical LPAR1, and LPAR3 all increased in the kid- analysis. *P,0.05; **P,0.01; ***P,0.001 versus vehicle group; n=6 in each group. ney, including in podocytes. This may

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Figure 7. Antagonism of LPARs slowed the loss of podocytes. (A) After treatment from 8 to 20 weeks of age, kidney mRNA levels of podocin and nephrin, two specific podocyte markers, were markedly higher in LPARI treated mice than in vehicle and RASI treated mice. **P,0.01 versus vehicle group; #P,0.05 versus RASI group; n=6 in each group. (B) Podocyte number per glomerulus was significantly higher in the LPARI group than vehicle and RASI groups when the mice were euthanized at 20 weeks of age. Bonferroni t tests were used for statistical analysis. Original magnification, 3400. ***P,0.001 versus vehicle at 20 weeks of age; ###P,0.001 versus RASI group at 20 weeks of age. explain why in our model, treatment with LPARI protected C57BLKS/J (BKS) background were generated as described previ- against podocyte loss. In addition, increased LPAR3 in glomer- ously.22 Genotyping was performed by PCR. At 8 weeks of age, the uli was validated in human diabetic kidneys. animals were divided into three groups (n=10 per group) and treated 2 2 We and others previously reported that in eNOS / mice, twice a day through gastric gavage with vehicle (0.5% Methocel E4M an acute podocyte injury occurred as early as 2 weeks after and 1.0% Soluplus in water), BMS002 at 20 mg/kg per day (dissolved in inducing diabetes with streptozotocin, and either captopril or vehicle), or a positive control (losartan plus enalapril, 10 mg/kg per day for losartan prevented podocytopathy.21 In this study, we treated each). The treatments lasted 12 weeks (from 8 to 20 weeks of age). 2 2 eNOS / db/db mice with enalapril and losartan at the age of BMS002 is an LPA1/3R dual antagonist. The functional antagonist affin- 8 weeks, when podocyte injury already existed. At 8–12 weeks ities (Kb) of BMS002 were assessed by its ability to block LPA-stimulated of age, podocyte pseudocysts, cytoplasmic vacuoles, and focal Ca2+ flux in CHO cells transfected with the appropriate human LPAR 6 areas of foot process effacement were already apparent in isoform. BMS002 demonstrated a Kb=7.6 nM ( 0.4 SEM) for LPAR1 2/2 6 eNOS db/db mice.ItispossiblethatRAASblockadeisef- and Kb=30.6 nM ( 2 SEM) for LPAR3, respectively. BMS002 was signif- 6 fective to prevent diabetic podocyte injury in this model, but less icantly less potent at LPAR2, with a Kb=290 nM ( 12 SEM). BMS002 effective to stop the progression of diabetic podocyte injury. was a full (100%) inhibitor of LPA-mediated Ca2+ flux at all three LPAR In summary, these studies indicate that in an accelerated isoforms. Losartan is an angiotensin II type 1 receptor antagonist and model of diabetic nephropathy, an intrarenal LPA/LPAR is enalapril is a prodrug that is bioactivated after oral administration by activated and mediates development of glomerulosclerosis hydrolysis of the ethyl ester to enalaprilat, an active ACEI. and tubulointerstitial fibrosis because antagonism of LPAR1/ LPAR3 markedly decreased the renal injury. Because LPAR Measurements of GFR in Conscious Mice antagonism did not significantly decrease the hypertension GFR was measured as we have reported previously.32 GFR was mea- 2 2 seen in eNOS / db/db mice and provided protection against sured before initiation of the experiment (7–8 weeks of age) and at the podocyte loss not seen with RAS inhibition, LPAR antagonism end of the experiment (20 weeks of age). might provide complementary beneficial effects to RAS inhibition to slow progression of diabetic nephropathy. Measurements of Urinary Albumin Excretion Urinary albumin and creatinine excretion was determined using Albuwell M kits (Exocell, Philadelphia, PA). Albuminuria is expressed CONCISE METHODS as urinary albumin concentration versus creatinine concentration ratio (micrograms per milligram). Animal Studies All animal experiments were performed in accordance with the Histologic Analysis guidelines of the Institutional Animal Care and Use Committee of Slides stained with periodic acid–Schiff were evaluated for glomerular 2 2 VanderbiltUniversity.Type2diabeticeNOS / db/db mice on the injury without knowledge of the identity of the various groups. A

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Figure 8. Antagonism of LPARs attenuated oxidative stress and preserved AKT2 activity. (A) 4-HNE, a biomarker of oxidative stress (detection of lipid peroxidation), was evident in kidneys (particular podocytes and other cell types in glomeruli) in vehicle-treated 2 2 eNOS / db/db mice, but its staining was minimal in LPARI-treated animals. Original magnification, 3250. (B) Renal AKT2 activity 2 2 (phosphorylated AKT) was marked lower in eNOS / db/db mice than in age- and sex-matched BKS controls. ***P,0.001; n=4 in BKS 2 2 2 2 controls and n=6 in eNOS / db/db mice. (C) AKT2 activity (phosphorylated AKT) was marked higher in LPARI-treated eNOS / db/db 2 2 mice than in vehicle-treated eNOS / db/db mice. *P,0.05; n=4 in vehicle group and n=6 in LPARI group. (D) Compared with 2 2 vehicle-treated eNOS / db/db mice, p-GSK3a (activated GSK3a) expression increased in podocytes with LPAR inhibition. Original magnification, 3400. semiquantitative index was used to evaluate the degree of glomerular (Ser21, no. 9316) were from Cell Signaling Technology; monoclonal sclerosis. Each glomerulus on a single section was graded from 0 to 4, mouse anti-4-HNE (no. 198960) was from R&D Systems. where 0 represents no lesion, and 1, 2, 3, and 4 represent sclerosis involving #25, 25–50, 50–75, or .75% of the glomerular tuft area, RNA Isolation and Quantitative RT-PCR respectively.15,33 Total renal RNAs were isolated using TRIzol Reagents (Invitrogen). Quantitative RT-PCR was performed using TaqMan real-time PCR Antibodies (7900HT; Applied Biosystems). The Master Mix and all gene probes Rat anti-mouse F4/80 (MCA497R) and CD3 (MCA1477) were pur- were also purchased from Applied Biosystems. The probes used in chased from AbD Serotec (now Bio-Rad); rabbit anti-fibronectin the experiments included mouse S18 (Mm02601778), IL-4Ra (F3648) and mouse anti–a-SMA (a marker of myofibroblasts, (Mm01275139), nephrin (Nphs1, Mm00497828), podocin (Nphs2, A5228) were from Sigma-Aldrich; rabbit anti-murine collagen type Mm01292252), CCL2 (MCP-1, Mm00441242), collagen I (Col1a1, I(600–401–103–01) was from Rockland Immunochemicals; rabbit Mm00801666), fibronectin 1 (Fn1, Mm01256744), a-SMA (acta2, anti-Wilms tumor protein (WT1, ab89901) and LPAR3 (ab23692, for Mm01546133), CTGF (Mm01192933), TGF-b (Mm00441726), F4/ immunoblotting) were from Abcam; rabbit anti-ENPP2/autotaxin 80 (Emr1, Mm00802529), CD3 (Cd3d, Mm00442746), 15-lipoxyge- (NBP1–32162), LPAR1 (NBP1–00788), and LPAR3 (NBP1–84903) nase (Alox15, Mm01250458), IL-23 (Il-23a, Mm00518984), IL-1a were from Novus Biologicals; goat anti-nephrin (AF3159) was from (Mm00439621), IL-1b (Mm00434228), IL-6 (Mm00446190), and R&D Systems; rabbit anti-Nitro tyrosine antibody (SC-55256) was INF-g (IFNg, Mm01168134). from Santa Cruz Biotechnology; rabbit anti-LPAR3 for immunoblotting was from Cayman Chemicals (10005280); rabbit anti-PERK Immunoblotting (Thr202/Tyr204, no. 4370), p-AKT (Thr308, no. 13038), p-ATK Kidney samples were homogenized with buffer containing 10 mM (Ser473, no. 4060), p-AKT2 (Ser474, no. 8599), and p-GSK3a TriszHCl(pH7.4),50mMNaCl,2mMEGTA,2mMEDTA,0.5%

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Figure 9. LPA signaling is activated in human diabetic kidney. (A) Double immunofluorescence staining with nephrin determined that autotaxin and LPAR3 were localized to podocytes in human diabetic kidneys. Original magnification, 3400. (B) LPAR1 was localized to tubular epithelia, with minimal expression in glomerulus in human diabetic kidneys. Original magnification, 3250.

Nonidet P-40, 0.1% SDS, 100 mMNa3VO4, 100 mM NaF, 0.5% and then incubated overnight at 4°C with goat anti-nephrin (R&D sodium deoxycholate, 10 mM sodium pyrophosphate, 1 mM Systems) plus rabbit anti-autotaxin, LPAR1, or LPAR3 diluted in in- PMSF, 10 mg/ml aprotinin, and 10 mg/ml leupeptin. The homogenate cubation buffer (PBS supplemented with 1% BSA, 1% normal donkey was centrifuged at 15,0003g for 20 minutes at 4°C. An aliquot of serum, 0.3% Triton X-100, and 0.01% azide). After washing with PBS, supernatant was taken for protein measurement with a BCA Protein the sections were incubated with incubation buffer containing Cy3 Assay kit (ThermoScientific, Rockford, IL). Immunoblotting was de- conjugated donkey anti-goat IgG and FITC conjugated anti-rabbit IgG scribed in a recent article.34 for 1 hour. After washing and staining with DAPI, sections were viewed and imaged with a Zeiss Axio fluorescence microscope and Immunofluorescence/Immunohistochemistry Staining spot-cam digital camera (Zeiss Microimaging, city, state/country). On and Quantitative Image Analysis the basis of the distinctive density and color of immunostaining in The animalswereanesthetizedwithpentobarbital(Nembutal,70mg/kg, video images, the number, size, and position of stained cells were administered intraperitoneally) and given heparin (1000 U/kg, ad- quantified using the BIOQUANT true-color windows system (R&M ministered intraperitoneally) to minimize coagulation. One kidney Biometrics) as previously described.36 Six representative fields from was removed for isolation of glomeruli. The other kidney was cut each animal were quantified, and their average was used as data from into four similar parts, one part for quantitative RT-PCR, one part for one animal sample. Podocyte density is expressed as podocytes per histologic analysis, one part stored at 280C for immunoblotting, glomerulus. and one part immersed in FPAS (3.7% formaldehyde, 10 mM sodium m-periodate, 40 mM phosphate buffer, and 1% acetic acid) for BP Measurement immunohistochemical and immunofluorescence staining. The fixed BP was measured in awake mice with a tail-cuff microphonic ma- kidney was dehydrated through a graded series of ethanols, embed- nometer.37 In brief, mice were trained for three consecutive days at ded in paraffin, sectioned (4 mm), and mounted on glass slides. room temperature (Monday to Wednesday) before SBP was recorded Immunostaining was carried out as previously described.35 For im- on the following 2 days (Thursday and Friday) using a tail-cuff mon- munofluorescence staining, deparaffinized sections were rehydrated, itor (BP-2000 BP Analysis System; Visitech Systems). SBPs recorded blocked with PBS containing 10% normal donkey serum for 1 hour, on two consecutive days were averaged and used as the SBP from one

J Am Soc Nephrol 28: 3300–3311, 2017 Lysophosphatidic Acid and Diabetic Nephropathy 3309 BASIC RESEARCH www.jasn.org mouse. BP was also measured in awake, catheterized mice in a subset REFERENCES of mice as previously described.37 1. Aikawa S, Hashimoto T, Kano K, Aoki J: Lysophosphatidic acid as a lipid mediator with multiple biological actions. J Biochem 157: 81–89, 2015 Podocyte Counting 2. Cholia RP, Nayyar H, Kumar R, Mantha AK: Understanding the multi- 38 Podocytes per glomerulus were estimated by the method of Weibel. faceted role of ectonucleotide pyrophosphatase/phosphodiesterase Briefly, 4 mm sections of formalin-fixed tissue were immunostained 2 (ENPP2) and its altered behaviour in human diseases. Curr Mol Med with WT-1 as a marker of podocyte nuclei. A total of 25 glomerular 15: 932–943, 2015 sections for each animal were photographed with a digital camera. 3. 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