Blocking lysophosphatidic acid receptor 1 signaling inhibits diabetic nephropathy in db/db mice  Hui Ying Li, Yoon Sin Oh, Ji-Woong Choi, Ji Yong Jung, Hee-Sook Jun  Kidney International  Volume 91, Issue 6, Pages 1362-1373 (June 2017) DOI: 10.1016/j.kint.2016.11.010 Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 1 mRNA and protein levels of lysophosphatidic acid receptor 1 (LPAR1) are upregulated in both the renal cortex of db/db mice and simian virus-transformed mouse mesangial (SV40 MES13) cells maintained in high glucose. mRNAs and proteins were isolated from the renal cortex of 16-week-old wild-type and db/db mice. (a) mRNA levels of autotaxin (ATX), LPAR1, LPAR2, and LPAR3 were measured by quantitative real-time polymerase chain reaction (qRT PCR) (n = 3–4). (b) Protein levels of ATX, LPAR1, LPAR2, and LPAR3 were measured by Western blot. (c) The results were quantified and β-actin was used as a loading control (n = 6–8). Representative pictures of (d) immunofluorescence staining for colocalization of α–smooth muscle actin (α-SMA) or Wilms' tumor 1 (WT-1) with LPAR1, which were examined in the kidney sections of 16-week-old db/db mice. Bars = 20 μm (n = 6). (e,f) mRNA and protein levels of LPAR1 and LPAR2 in SV40 MES13 cells maintained in low glucose (5 mM), low glucose (5 mM) + mannitol (19.44 mM), and high glucose (25 mM) were examined by qRT PCR and Western blot. (g) The results were quantified, and β-actin was used as a loading control (n = 3 independent experiments). *P < 0.05. Data represent the mean ± SEM. DAPI, 4′,6-diamidino-2-phenylindole. Kidney International 2017 91, 1362-1373DOI: (10.1016/j.kint.2016.11.010) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 2 Treatment with ki16425 ameliorates mesangial matrix accumulation in cortex of db/db mice. Eight-week-old wild-type and db/db mice were injected with vehicle or ki16425 (10 mg/kg i.p.) for 8 weeks, and mice were killed. (a) Representative staining pictures of hematoxylin and eosin (H & E), periodic acid–Schiff (PAS), and fibronectin staining of kidney tissues in mice. Bars = 20 μm. (b) Glomerular tuft area, (c) glomerular volume, and (d) the mesangial matrix index (percentage) were quantified from PAS staining using ImageJ Software (30 glomeruli per mouse, n = 6). *P < 0.05. Data represent mean ± SEM. Kidney International 2017 91, 1362-1373DOI: (10.1016/j.kint.2016.11.010) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 3 Treatment with ki16425 reduces the expression of transforming growth factor (TGF-β) in both the renal cortex of db/db mice and lysophosphatidic acid (LPA)-treated simian virus-transformed mouse mesangial (SV40 MES13) cells. Mice were treated as described in Figure 2. (a) mRNA expression of TGF-β in the renal cortex was determined by quantitative real-time polymerase chain reaction (qRT PCR) (n = 9 to 10). (b) Representative Western blots of TGF-β protein expression. (c) The results were quantified, and β-actin was used as a loading control (n = 9 to 10). (d) Representative pictures of immunofluorescence staining for phospho-Smad2/3 protein expression. Bars = 20 μm (n = 6). (e−g) SV40 MES13 cells were treated with 10 μM of LPA for the indicated times. (e) TGF-β mRNA levels were analyzed by qRT PCR. (f) TGF-β protein levels were analyzed by Western blot. (g) The results were quantified, and β-actin was used as loading control (n = 3 independent experiments). (h−j) SV40 MES13 cells were treated with LPA (10 μM) with or without ki16425 (10 μM) for 3 hours. (h) TGF-β mRNA levels were analyzed by qRT PCR. (i) Protein levels were analyzed by Western blot. (j) The results were quantified, and β-actin was used as a loading control. (k) Normal human mesangial cells were treated with LPA (10 μM) with or without ki16425 (10 μM) for 1.5 hours. TGF-β protein levels were analyzed by Western blot (n = 3 independent experiments). (l) TGF-β siRNA and scrambled siRNA (sicon) were transfected into SV40 MES13 cells, and TGF-β protein levels were analyzed by Western blot (n = 3 independent experiments). (m) LPA-induced fibronectin protein expression was examined by Western blot analysis at 12 hours after treatment in cells with TGF-β siRNA or sicon (n = 3 independent experiments). *P < 0.05. Data represent mean ± SEM. Kidney International 2017 91, 1362-1373DOI: (10.1016/j.kint.2016.11.010) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 4 Knockdown of lysophosphatidic acid receptor 1 (LPAR1) decreases LPA-induced transforming growth factor−β (TGF-β) expression and overexpression of LPAR1 upregulates LPA-induced TGF-β expression in simian virus-transformed mouse mesangial (SV40 MES13) cells. (a) LPAR1 siRNA or scrambled siRNA (sicon) was transfected into SV40 MES13 cells, and LPAR1 protein levels were analyzed by Western blot (n = 3 independent experiments). (b) LPA-induced TGF-β protein expression was examined by Western blot analysis at 6 hours after LPA treatment in cells treated with LPAR1 siRNA or sicon (n = 3 independent experiments). (c) pEGFP-LPAR1 (LPAR1) or an empty vector (vector) was transfected into SV40 MES13 cells, and LPAR1 mRNA levels were analyzed by quantitative real-time polymerase chain reaction (n = 3 independent experiments). (d) Protein level of LPAR1 was measured by Western blot (n = 3 independent experiments). (e) LPA-induced TGF-β was examined at 6 hours after LPA treatment in cells overexpressing LPAR1 and analyzed by Western blot. *P < 0.05. Data represent mean ± SEM. Kidney International 2017 91, 1362-1373DOI: (10.1016/j.kint.2016.11.010) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 5 Lysophosphatidic acid (LPA) phosphorylates glycogen synthase kinase 3β (GSK3β) at Ser9 and induces nuclear mature SREBP1 (m-SREBP1) accumulation in simian virus-transformed mouse mesangial (SV40 MES13) cells. (a) GSK3β, phospho-GSK3β (Ser9), and (c) m-SREBP1 and Lamin B expression after 10 μM of LPA treatment was analyzed by Western blot. (b,d) The results were quantified, and β-actin and Lamin B were used as loading controls (n = 3 independent experiments). *P < 0.05. Data represent mean ± SEM. Kidney International 2017 91, 1362-1373DOI: (10.1016/j.kint.2016.11.010) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 6 Glycogen synthase kinase 3β (GSK3β) (Ser9) phosphorylation and SREBP1 activation are involved in lysophosphatidic acid (LPA)-induced transforming growth factor−β (TGF-β) expression in simian virus-transformed mouse mesangial (SV40 MES13) cells. (a−c) SV40 MES13 cells were pretreated with PP2 (20 μM) for 1 hour, then treated with LPA (10 μM) for 1 hour. The total cell lysate and nuclear fraction were extracted. (a) GSK3β, phospho-GSK3β (Ser9), and mature (m)-SREBP1 levels were analyzed by Western blot. (b,c) The results were quantified and Lamin B was used as a loading control (n = 3 independent experiments). (d) HA-GSK3β-S9A (constitutively active GSK3β) and empty vector (vector) were transfected into SV40 MES13 cells, and HA protein levels were analyzed by Western blot (n = 3 independent experiments). (e) After transfection with HA-GSK3β-S9A or control vector in SV40 MES13 cells, cells were treated LPA (10 μM) for 1 hour with or without MG132 (10 μM). The nuclear fraction was extracted, and m-SREBP1 levels were analyzed by Western blot. (f) The results were quantified, and lamin B was used as a loading control (n = 3 independent experiments). (g) Cells were treated with LPA (10 μM) for 1 hour with or without preincubation with fatostatin (20 μM) for 4 hours. The nuclear fraction was extracted, and m-SREBP1 levels were analyzed by Western blot. (h) The results were quantified, and Lamin B was used as a loading control (n = 3 independent experiments). (i,j) Cells were treated with LPA (10 μM) for 3 hours (mRNA) and 6 hours (protein) with or without preincubation with fatostatin (20 μM) for 4 hours; the mRNA and protein levels of TGF-β were analyzed by quantitative real-time polymerase chain reaction and Western blot. (k) The results were quantified, and β-actin was used as a loading control (n = 3 independent experiments). *P < 0.05. Data represent mean ± SEM. PP2, kinase inhibitor (4-amino-5-[4-chlorophenyl]-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine). Kidney International 2017 91, 1362-1373DOI: (10.1016/j.kint.2016.11.010) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 7 Treatment with ki16425 inhibits glycogen synthase kinase 3β (GSK3β) (Ser9) phosphorylation and mature (m)-SREBP1 nuclear accumulation in the renal cortex of db/db mice. Mice were treated as described in Figure 2. (a) Representative blots of GSK3β, phospho-GSK3β (Ser9) of whole renal cortical tissue, and (c) m-SREBP1 in nuclear fractions of renal cortical tissue, which were analyzed by Western blot. (b,d) The results were quantified, and β-actin and Lamin B were used as loading controls (n = 9 to 10). *P < 0.05. Data represent mean ± SEM. Kidney International 2017 91, 1362-1373DOI: (10.1016/j.kint.2016.11.010) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 8 Schematic diagram showing the pathway involved in lysophosphatidic acid (LPA)–induced transforming growth factor–β (TGF-β) synthesis and glomerular injury, which is mediated by phospho–glycogen synthase kinase 3β (GSK3β) (Ser9) and SREBP1 activation. SCAP, SREBP cleavage-activating protein. Kidney International 2017 91, 1362-1373DOI: (10.1016/j.kint.2016.11.010) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure S1 Expression level of lipid phosphate phosphatases (LPPs) is not changed in the kidney cortex of db/db mice compared with wild-type mice. mRNAs were isolated from the renal cortex of 16-week-old wild-type and db/db mice, and mRNA levels of (A) LPP1, (B) LPP2, and (C) LPP3 were measured by q-real-time polymerase chain reaction (q-RT PCR) (n = 3). Data are expressed as the ratio relative to the levels found in wild-type mice. Kidney International 2017 91, 1362-1373DOI: (10.1016/j.kint.2016.11.010) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure S2 LPAR1 expression is increased in α–smooth muscle actin (α-SMA)−positive cells of db/db mice. Representative pictures of immunofluorescence staining for colocalization of (A) α-SMA or (B) SMA with LPAR1, which were examined in the kidney sections of 16-week-old wild-type mice and db/db mice. Bars = 20 μm (n = 6). Kidney International 2017 91, 1362-1373DOI: (10.1016/j.kint.2016.11.010) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure S3 Treatment with ki16425 does not affect the glucose tolerance and insulin sensitivity of db/db mice. Eight-week-old wild-type and db/db mice were injected i.p. with vehicle or ki16425 (10 mg/kg) for 8 weeks. (A) For glucose tolerance tests, mice fasted overnight and were then injected with glucose (2 g/kg i.p.). A glucometer (Onetouch; Lifescan, Milpitas, CA) was used to measure glucose in the tail vein blood at 0, 30, 60, 120, and 150 minutes after injection. (B) For insulin tolerance tests, mice were injected with insulin (2 U/kg, i.p.; Green Cross, Yongin, Korea), and tail vein blood glucose was measured at 0, 30, 60, and 90 minutes after injection. Kidney International 2017 91, 1362-1373DOI: (10.1016/j.kint.2016.11.010) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure S4 Treatment with ki16425, but not H2L5186303, reduces the expression of fibronectin in the renal cortex of db/db mice or lysophosphatidic acid (LPA)-treated simian virus-transformed mouse mesangial (SV40 MES13) cells. Eight-week-old wild-type and db/db mice were injected with vehicle or ki16425 (10 mg/kg i.p.) for 8 weeks. (A) The mRNA level of fibronectin in the renal cortex was determined by quantitative real-time polymerase chain reaction (qRT PCR) (n = 9 to 10). (B) SV40 MES 13 cells were treated with various concentrations of LPA for 24 hours, and the mRNA level of fibronectin was analyzed by qRT PCR (n = 3 independent experiments). (C) SV40 MES 13 cells were treated with LPA (10 μM) for the indicated times, and mRNA level of fibronectin was analyzed by qRT PCR (n = 3 independent experiments). (D) Cells were treated with LPA (10 μM) without (con) or with ki16425 (10 μM) for 3 hours, and fibronectin mRNA levels were analyzed by qRT PCR (n = 3 independent experiments). (E and F) Cells were treated with LPA (10 μM) with or without various concentrations of H2L5186303 for 3 hours, and (E) transforming growth factor-β (TGF-β) and (F) fibronectin mRNA levels were analyzed by qRT PCR (n = 3 independent experiments). *P < 0.05. Data represent mean ± SEM. Kidney International 2017 91, 1362-1373DOI: (10.1016/j.kint.2016.11.010) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure S5 AKT/PI3K pathway is involved in lysophosphatidic acid (LPA)-induced transforming growth factor-β (TGF-β) expression in simian virus-transformed mouse mesangial (SV40 MES13 cells). SV40 MES13 cells were treated with LPA (10 μM) for 6 hours with or without preincubation with LY294002 (20 μM) for 30 minutes. TGF-β protein expression was analyzed by Western blot (n = 3 independent experiments, representative blot shown). Kidney International 2017 91, 1362-1373DOI: (10.1016/j.kint.2016.11.010) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure S6 Exogenous transforming growth factor-β (TGF-β) increases the protein expression level of autotaxin (ATX) in simian virus-transformed mouse mesangial (SV40 MES) cells. (A) SV40 MES13 cells were treated with 5 ng/ml of TGF-β for 12 hours and ATX protein expression was analyzed by Western blot. (B) The results were quantified and β-actin was used as loading control (n = 3 independent experiments). Kidney International 2017 91, 1362-1373DOI: (10.1016/j.kint.2016.11.010) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure S7 Lysophosphatidic acid (LPA) activates the mitogen-activated protein kinase (MAPK) pathway in simian virus-transformed mouse mesangial (SV40 MES) cells. SV40 MES13 cells were treated with 10 μM of LPA for the indicated times. Phospho-ERK, phospho-JNK, phospho-p38, and β-actin expression were analyzed by Western blot (n = 3 independent experiments; representative blot shown). Kidney International 2017 91, 1362-1373DOI: (10.1016/j.kint.2016.11.010) Copyright © 2016 International Society of Nephrology Terms and Conditions