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Background: SGLT2 inhibitor (SGLT2i) displayed remarkable and early renal protection in overt nephropathy cases in clinical trials. However, detailed molecular mechanisms by which SGLT2i ameliorated renal damage in proteinuric diabetic patients were not elucidated yet. Here, we investigated that SGLT2i exerted renal protective effects via adenosine signal in diabetic mice with overt proteinuria. Methods: STZ (200mg/kgBW) was injected intraperitoneally to induce diabetes in 8-week-old CD-1 mice. Four weeks later, mice were treated with intraperitoneal injection of bovine serum albumin (BSA: 0.3g/30gBW) with or without oral administration of SGLT2i TA-1887 (30mg/kg). Result: In the STZ+BSA, pronounced renal tubular damages and interstitial fibrosis were observed; TA-1887 significantly ameliorated. The mRNA microarray analysis of kidney samples revealed that gene expressions related to TGF-β signaling pathway and epithelial-mesenchymal-transition program were increased in the STZ+BSA compared to the Control+BSA; TA-1887 suppressed them. Furthermore, the expression of ecto 5'-nucleotidase (5'-NT), which is essential for production of adenosine from AMP, was suppressed in STZ+BSA compared to Control+BSA; TA-1887 restored 5’-NT levels. In the immunohistological analysis, 5'-NT was expressed in glomeruli including mesangial cells and renal tubules of cortex/medulla in control kidney. However, 5’-NT was suppressed in all areas of STZ+BSA kidney; TA-1887 reversed the 5'-NT expression and distribution. Glomerular filtration rate (GFR) evaluated by inulin clearance was significantly greater in the STZ+BSA group and suppressed by TA-1887; the administration of the adenosine type 1 receptor inhibitor, DPCPX, cancelled the effects of TA-1887 on GFR and renal tubule damage. Conclusion: The adenosine signal plays an important role in the renal protective effects of SGLT2i in diabetic mice with proteinuria. Disclosure K. Shimada: None. Y. Takagaki: None. K. Kanasaki: Other Relationship; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Mitsubishi Tanabe Pharma Corporation, Ono Pharmaceutical Co., Ltd., Sanofi, Taisho Toyama Pharmaceutical. D. Koya: Research Support; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Mitsubishi Tanabe Pharma Corporation. Speaker's Bureau; Self; Astellas Pharma Inc., Astellas Pharma Inc., AstraZeneca, Merck & Co., Inc., Mitsubishi Tanabe Pharma Corporation, Taisho Pharmaceutical Co., Ltd. Funding Japan Society for the Promotion of Science

Background: In diabetic patients, the incident of heart failure is two to five times greater when compared to that of general population. The influence of DPP-4 inhibitor on the heart failure and cardiac damage in diabetes is not completely elucidated yet. Therefore, to understand the pathobiology of heart damage in diabetes and the influence of DPP-4 inhibitor is emerged as significant. Necroptosis, recently identified as programmed cell death, had shown to be involved in heart damage of ischemic heart disease model. Here we found that intervention with DPP-4 inhibitor linagliptin (LINA) ameliorated heart damage in diabetic mice associated with the suppression of necroptosis. Method: We utilized streptozotocin-induced diabetic CD-1 mice (STZ:200mg/kg). At 20 weeks after the onset of diabetes, diabetic mice were treated with LINA (STZ+LINA) for 4 weeks. Result: Blood pressure, blood glucose, body and heart weights were not different between STZ and STZ+LINA. Ultrasonic Echocardiography analysis revealed that LVDd and LVDs were significantly decreased in STZ and STZ+LINA compared with control. STZ-induced diabetic CD-1 mice exhibited perivascular fibrosis evaluated by picrosirius red staining and strong immunoreactivity for DPP-4 expression in cardiac interstitium; STZ+LINA exhibited suppression of DPP-4 and amelioration of heart fibrosis. Diabetic CD-1 mice exhibited deficiency in myocardial stripe structure and endothelial damage in electron microscopy; LINA normalized all. When necroptosis was evaluated by associated kinases, RIP3 and MLKL, diabetic CD-1 mice were exhibited a strong RIP3 and MLKL mRNA and protein expression levels in myocyte; LINA suppressed RIP3 and MLKL levels in myocyte. Conclusion: These results indicated that LINA restored cardiac structure associated with suppression of necroptosis in type 1 diabetic mice. Disclosure T. Hirai: None. Y. Takagaki: None. K. Kanasaki: Other Relationship; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Mitsubishi Tanabe Pharma Corporation, Ono Pharmaceutical Co., Ltd., Sanofi, Taisho Toyama Pharmaceutical. D. Koya: Research Support; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Mitsubishi Tanabe Pharma Corporation. Speaker's Bureau; Self; Astellas Pharma Inc., Astellas Pharma Inc., AstraZeneca, Merck & Co., Inc., Mitsubishi Tanabe Pharma Corporation, Taisho Pharmaceutical Co., Ltd. Funding Japan Society for the Promotion of Science

Kidney fibrosis is the final common pathway of progressive kidney disease including diabetic kidney disease (DKD). With the rapid increase of type 2 diabetes, DKD in type 2 diabetic patients has become a serious public health threat around the world. To establish novel therapy to combat DKD, human disease relevant animal model is essential. However, a type 2 diabetic mouse model with presenting progressive kidney fibrosis is not established yet. We have previously reported that kidneys of streptozotocin-induced diabetic CD-1 mice exhibited severe fibrosis compared to other backgrounds of mice associated with the suppression of anti-fibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline, fibroblast growth factor receptor 1, and their downstream target anti-fibrotic microRNAs (miRs) such as miR 29 and miR let-7. As the type 2 diabetic mice model, leptin receptor deficient mice in BKS background (BKSdb/db) are often utilized; the kidney fibrosis in BKSdb/db phenotype is minimum. Here we backcrossed db gene into potential fibrotic CD-1 background and found that male CD-1db/db exhibited severe kidney fibrosis when compared to male BKSdb/db. We have already performed the backcrosses for 11 generations. Body weight of CD-1db/db was comparable but drank up to 3 times more water compared to BKSdb/db. Blood glucose levels in CD-1db/db appeared to be higher when compared to BKSdb/db. When evaluated the per body weight ratio, kidney and heart weighed no significant differences between CD-1db/db and BKSdb/db. CD-1db/db exhibited severe tubulo-interstitial fibrosis, glomerulosclerosis, and tubular epithelial cells damage. Heart in CD-1db/db displayed fibrotic change as well. In conclusion, here we provide novel fibrotic type 2 DKD mouse model. CD-1db/db could be excellent model to perform experimental therapy for DKD. Disclosure Y. Mizunuma: None. K. Nitta: None. S. Takagi: None. K. Kanasaki: Advisory Panel; Self; Boehringer Ingelheim Pharmaceuticals, Inc.. Speaker's Bureau; Self; Eli Lilly and Company, Sanofi, Mitsubishi Tanabe Pharma Corporation. Research Support; Self; Boehringer Ingelheim GmbH. Speaker's Bureau; Self; Boehringer Ingelheim GmbH, Astellas, Astellas. Research Support; Self; Ono Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Corporation. D. Koya: None.

Kidney fibrosis is the final common pathway of all progressive chronic kidney diseases, of which diabetic nephropathy is the leading cause. Endothelial-to-mesenchymal transition (EndMT) has emerged as one of the most important origins of matrix-producing fibroblasts. Dipeptidyl peptidase-4 (DPP-4) inhibitors have been introduced into the market as antidiabetes drugs. Here, we found that the DPP-4 inhibitor linagliptin ameliorated kidney fibrosis in diabetic mice without altering the blood glucose levels associated with the inhibition of EndMT and the restoration of microRNA 29s. Streptozotocin-induced diabetic CD-1 mice exhibited kidney fibrosis and strong immunoreactivity for DPP-4 by 24 weeks after the onset of diabetes. At 20 weeks after the onset of diabetes, mice were treated with linagliptin for 4 weeks. Linagliptin-treated diabetic mice exhibited a suppression of DPP-4 activity/protein expression and an amelioration of kidney fibrosis associated with the inhibition of EndMT. The therapeutic effects of linagliptin on diabetic kidneys were associated with the suppression of profibrotic programs, as assessed by mRNA microarray analysis. We found that the induction of DPP-4 observed in diabetic kidneys may be associated with suppressed levels of microRNA 29s in diabetic mice; linagliptin restored microRNA 29s and suppressed DPP-4 protein levels. Using cultured endothelial cells, we found that linagliptin inhibited TGF-β2–induced EndMT, and such anti-EndMT effects of linagliptin were mediated through microRNA 29 induction. These results indicate the possible novel pleiotropic action of linagliptin to restore normal kidney function in diabetic patients with renal impairment.

We have previously reported that N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), which is a tetrapeptide hydrolyzed by ACE, inhibits the transforming growth factor-β (TGF-β)-induced expression of extracellular matrix proteins via inhibition of the Smad signaling in human mesangial cells. To test in vivo the antifibrotic efficacy of Ac-SDKP, we examined whether long-term Ac-SDKP treatment can prevent renal insufficiency and glomerulosclerosis in diabetic db/db mice. Diabetic db/db mice or nondiabetic db/m mice were treated with Ac-SDKP for 8 weeks using osmotic minipumps. The treatment with Ac-SDKP increased plasma Ac-SDKP concentrations by approximately threefold in both groups but did not affect the blood glucose levels. Histologically, the increased glomerular surface area, mesangial matrix expansion, and overproduction of extracellular matrix proteins in db/db mice were significantly inhibited by Ac-SDKP. Furthermore, Ac-SDKP treatment normalized the increased plasma creatinine value in db/db mice, whereas the albuminuria in Ac-SDKP–treated db/db mice was somewhat decreased as compared with nontreated db/db mice, although the difference was not statistically significant. In addition, the nuclear translocation of Smad3 was inhibited by Ac-SDKP. These results demonstrate that long-term Ac-SDKP treatment ameliorates renal insufficiency and glomerulosclerosis in db/db mice via inhibition of TGF-β/Smad pathway, suggesting that Ac-SDKP could be useful in the treatment of diabetic nephropathy.