Diabetes Metab J.  2016 Jun;40(3):211-221. 10.4093/dmj.2016.40.3.211.

Renoprotective Effect of Gemigliptin, a Dipeptidyl Peptidase-4 Inhibitor, in Streptozotocin-Induced Type 1 Diabetic Mice

• Jung GS
• Jeon JH
• Choe MS
• Kim SW
• Lee IK
• Kim MK
• Park KG

• KMID: 2308629
• DOI: http://doi.org/10.4093/dmj.2016.40.3.211

Abstract

BACKGROUND
Dipeptidyl peptidase-4 (DPP-4) inhibitors are widely used in the treatment of patients with type 2 diabetes and have proven protective effects on diabetic kidney disease (DKD). Whether DPP-4 inhibitors have renoprotective effects on insulin-deficient type 1 diabetes has not been comprehensively examined. The aim of this study was to determine whether gemigliptin, a new DPP-4 inhibitor, has renoprotective effects in streptozotocin (STZ)-induced type 1 diabetic mice.
METHODS
Diabetes was induced by intraperitoneal administration of a single dose of STZ. Mice with diabetes were treated without or with gemigliptin (300 mg/kg) for 8 weeks. Morphological changes of the glomerular basement membrane (GBM) were observed by electron microscopy and periodic-acid Schiff staining. In addition, we measured blood glucose and urinary albumin excretion and evaluated fibrotic markers using immunohistochemical staining, quantitative reverse transcription polymerase chain reaction analysis, and Western blot analysis.
RESULTS
Gemigliptin did not reduce the blood glucose levels of STZ-treated mice. In gemigliptin-treated mice with STZ, a significant reduction in urinary albumin excretion and GBM thickness was observed. Immunohistological examination revealed that gemigliptin attenuated renal fibrosis induced by STZ and decreased extracellular matrix protein levels, including those of type I collagen and fibronectin, and Smad3 phosphorylation. In cultured rat renal cells, gemigliptin inhibited transforming growth factor β-stimulated type I collagen and fibronectin mRNA and protein levels via down-regulation of Smad3 phosphorylation.
CONCLUSION
Our data demonstrate that gemigliptin has renoprotective effects on DKD, regardless of its glucose-lowering effect, suggesting that it could be used to prevent DKD, including in patients with type 1 diabetes.

Keyword

Diabetes mellitus, type 1; DPP-4 inhibitor; Renal fibrosis

MeSH Terms

Animals
Blood Glucose
Blotting, Western
Collagen Type I
Diabetes Mellitus, Type 1
Diabetic Nephropathies
Down-Regulation
Extracellular Matrix
Fibronectins
Fibrosis
Glomerular Basement Membrane
Humans
Mice*
Microscopy, Electron
Phosphorylation
Polymerase Chain Reaction
Rats
Reverse Transcription
RNA, Messenger
Streptozocin
Transforming Growth Factors
Blood Glucose
Collagen Type I
Fibronectins
RNA, Messenger
Streptozocin
Transforming Growth Factors

Figure

• 

  Fig. 1 Effect of gemigliptin on metabolic parameters. Diabetes was induced by intraperitoneal administration of a single dose of streptozotocin (STZ; 150 mg/kg/body weight). Diabetic mice were treated with or without an oral dose of gemigliptin (Gemi; 300 mg/kg/day) for 8 weeks (n=6). (A) Plasma glucagon-like peptide-1 (GLP-1) levels, (B) blood glucose levels, (C) body weight, (D) kidney weight-to-body weight ratio, urine albumin excretion (UAE), and urine albumin-to-creatinine ratio (UACR) were determined. CON, control. aP<0.001 compared with CON mice, bP<0.05, cP<0.001 compared with STZ-treated mice.

• 

  Fig. 2 Effect of gemigliptin (Gemi) on glomerular basement membrane (GBM) thickening. Electron microscopy of kidney sections (A), and periodic acid Schiff (PAS) staining (B) from control (CON) mice and streptozotocin (STZ)-induced diabetic mice, without or with Gemi treatment (300 mg/kg; STZ+Gemi; n=6). The arrow indicates GBM. Bar indicates 500 nm. Bar graph shows the changes in GBM thickness (A) and PAS-positive mesangial area (%) at week 8. aP<0.001, bP<0.01 compared with CON mice, cP<0.001 compared with STZ-treated mice.

• 

  Fig. 3 Effect of gemigliptin (Gemi) on streptozotocin (STZ)-induced renopathological changes. Representative images of renal sections from control (CON) mice, STZ-induced diabetic mice, without or with Gemi treatment (300 mg/kg; STZ+Gemi). The sections were (A) stained with H&E or Sirius red, or were (B) immunostained with antibodies targeting type I collagen and fibronectin. The number of atrophic tubules was determined by measuring abnormal and dilated tubular basement membranes in five random fields of H&E-stained sections under high power magnification (×200). Areas of positive staining with Sirius red, type I collagen, or fibronectin antibodies were quantified by computer-based quantitative morphometric analysis. All data were normalized to the CON (n=1) and expressed as the mean±SEM of five random fields of each kidney section (n=6 in each group). The effect of Gemi on type I collagen and fibronectin mRNA levels (C) and protein expression (D) were further examined by real-time reverse transcription polymerase chain reaction and Western blot analysis. aP<0.001, bP<0.01 compared with CON mice, cP<0.01, dP<0.001 compared with STZ-treated mice.

• 

  Fig. 4 Effects of gemigliptin (Gemi) on renal fibrosis gene expression in streptozotocin (STZ)-induced type 1 diabetic mice. (A) Representative images of renal sections from control (CON) mice, STZ-induced diabetic mice, without STZ or with Gemi treatment (300 mg/kg; STZ+Gemi). The sections were immunostained with antibodies targeting transforming growth factor β (TGF-β) and p-Smad3. Areas of positive staining were quantified by computer-based morphometric analysis. All data were normalized to the CON (n=1) and are represented as the mean±SEM of five random fields of each kidney section (n=6 in each group). (B) Representative Western blot analysis of renal protein expression levels of TGF-β and p-Smad3. The protein expression levels were normalized to those of β-tubulin. The data are represented as the mean±SEM of three independent measurements (n=6 in each group). aP<0.001, bP<0.01 compared with CON mice, cP<0.001 compared with STZ-treated mice.

• 

  Fig. 5 Effects of gemigliptin (Gemi) on transforming growth factor β (TGF-β)-stimulated p-Smad3, type I collagen, and fibronectin expression in cultured renal cells. (A) Representative real-time reverse transcription polymerase chain reaction (RT-PCR) of the expression levels of type I collagen and fibronectin in TGF-β-stimulated NRK-52E cells. (B) Representative Western blot analyses of the expression of p-Smad3, type I collagen, and fibronectin in TGF-β-stimulated NRK-52E cells. (C) Quantification of Western blot analyses in TGF-β-stimulated NRK-52E cells. (D) Representative real-time RT-PCR of the expression levels of type I collagen and fibronectin in TGF-β-stimulated rat mesangial cells (RMCs). (E) Representative Western blot analyses of the expression of p-Smad3, type I collagen, and fibronectin in TGF-β-stimulated RMCs. (F) Quantification of Western blot analyses of TGF-β-stimulated RMCs. Expression levels of mRNA were normalized to those of glyceraldehyde 3-phosphate dehydrogenase, and protein expression levels were normalized to those of β-tubulin. The data are represented as the mean±SEM of three independent measurements (n=6 in each group). aP<0.01, bP<0.05, cP<0.001 compared with control mice, dP<0.01, eP<0.05, fP<0.001 compared with TGF-β alone.

Reference

1. Packham DK, Alves TP, Dwyer JP, Atkins R, de Zeeuw D, Cooper M, Shahinfar S, Lewis JB, Lambers Heerspink HJ. Relative incidence of ESRD versus cardiovascular mortality in proteinuric type 2 diabetes and nephropathy: results from the DIAMETRIC (Diabetes Mellitus Treatment for Renal Insufficiency Consortium) database. Am J Kidney Dis. 2012; 59:75–83.

2. Park CW. Diabetic kidney disease: from epidemiology to clinical perspectives. Diabetes Metab J. 2014; 38:252–260.

3. Muskiet MH, Smits MM, Morsink LM, Diamant M. The gut-renal axis: do incretin-based agents confer renoprotection in diabetes? Nat Rev Nephrol. 2014; 10:88–103.

4. Lan HY, Chung AC. TGF-β/Smad signaling in kidney disease. Semin Nephrol. 2012; 32:236–243.

5. Park CW, Kim HW, Ko SH, Lim JH, Ryu GR, Chung HW, Han SW, Shin SJ, Bang BK, Breyer MD, Chang YS. Long-term treatment of glucagon-like peptide-1 analog exendin-4 ameliorates diabetic nephropathy through improving metabolic anomalies in db/db mice. J Am Soc Nephrol. 2007; 18:1227–1238.

6. Kodera R, Shikata K, Kataoka HU, Takatsuka T, Miyamoto S, Sasaki M, Kajitani N, Nishishita S, Sarai K, Hirota D, Sato C, Ogawa D, Makino H. Glucagon-like peptide-1 receptor agonist ameliorates renal injury through its anti-inflammatory action without lowering blood glucose level in a rat model of type 1 diabetes. Diabetologia. 2011; 54:965–978.

7. Hendarto H, Inoguchi T, Maeda Y, Ikeda N, Zheng J, Takei R, Yokomizo H, Hirata E, Sonoda N, Takayanagi R. GLP-1 analog liraglutide protects against oxidative stress and albuminuria in streptozotocin-induced diabetic rats via protein kinase A-mediated inhibition of renal NAD(P)H oxidases. Metabolism. 2012; 61:1422–1434.

8. Mentlein R. Dipeptidyl-peptidase IV (CD26): role in the inactivation of regulatory peptides. Regul Pept. 1999; 85:9–24.

9. Weber AE. Dipeptidyl peptidase IV inhibitors for the treatment of diabetes. J Med Chem. 2004; 47:4135–4141.

10. Deacon CF, Holst JJ. Dipeptidyl peptidase IV inhibition as an approach to the treatment and prevention of type 2 diabetes: a historical perspective. Biochem Biophys Res Commun. 2002; 294:1–4.

11. Cernea S, Raz I. Therapy in the early stage: incretins. Diabetes Care. 2011; 34:Suppl 2. S264–S271.

12. Chrysant SG, Chrysant GS. Clinical implications of cardiovascular preventing pleiotropic effects of dipeptidyl peptidase-4 inhibitors. Am J Cardiol. 2012; 109:1681–1685.

13. Min HS, Kim JE, Lee MH, Song HK, Kang YS, Lee MJ, Lee JE, Kim HW, Cha JJ, Chung YY, Hyun YY, Han JY, Cha DR. Dipeptidyl peptidase IV inhibitor protects against renal interstitial fibrosis in a mouse model of ureteral obstruction. Lab Invest. 2014; 94:598–607.

14. Kodera R, Shikata K, Takatsuka T, Oda K, Miyamoto S, Kajitani N, Hirota D, Ono T, Usui HK, Makino H. Dipeptidyl peptidase-4 inhibitor ameliorates early renal injury through its anti-inflammatory action in a rat model of type 1 diabetes. Biochem Biophys Res Commun. 2014; 443:828–833.

15. Liu WJ, Xie SH, Liu YN, Kim W, Jin HY, Park SK, Shao YM, Park TS. Dipeptidyl peptidase IV inhibitor attenuates kidney injury in streptozotocin-induced diabetic rats. J Pharmacol Exp Ther. 2012; 340:248–255.

16. Kanasaki K, Shi S, Kanasaki M, He J, Nagai T, Nakamura Y, Ishigaki Y, Kitada M, Srivastava SP, Koya D. Linagliptin-mediated DPP-4 inhibition ameliorates kidney fibrosis in streptozotocin-induced diabetic mice by inhibiting endothelial-to-mesenchymal transition in a therapeutic regimen. Diabetes. 2014; 63:2120–2131.

17. Scirica BM, Bhatt DL, Braunwald E, Steg PG, Davidson J, Hirshberg B, Ohman P, Frederich R, Wiviott SD, Hoffman EB, Cavender MA, Udell JA, Desai NR, Mosenzon O, McGuire DK, Ray KK, Leiter LA, Raz I. SAVOR-TIMI 53 Steering Committee and Investigators. Saxagliptin and cardiovascular outcomes in patients with type 2 diabetes mellitus. N Engl J Med. 2013; 369:1317–1326.

18. White WB, Cannon CP, Heller SR, Nissen SE, Bergenstal RM, Bakris GL, Perez AT, Fleck PR, Mehta CR, Kupfer S, Wilson C, Cushman WC, Zannad F. EXAMINE Investigators. Alogliptin after acute coronary syndrome in patients with type 2 diabetes. N Engl J Med. 2013; 369:1327–1335.

19. Mori H, Okada Y, Arao T, Tanaka Y. Sitagliptin improves albuminuria in patients with type 2 diabetes mellitus. J Diabetes Investig. 2014; 5:313–319.

20. Fujita H, Taniai H, Murayama H, Ohshiro H, Hayashi H, Sato S, Kikuchi N, Komatsu T, Komatsu K, Komatsu K, Narita T, Yamada Y. DPP-4 inhibition with alogliptin on top of angiotensin II type 1 receptor blockade ameliorates albuminuria via up-regulation of SDF-1α in type 2 diabetic patients with incipient nephropathy. Endocr J. 2014; 61:159–166.

21. Kim SH, Lee SH, Yim HJ. Gemigliptin, a novel dipeptidyl peptidase 4 inhibitor: first new anti-diabetic drug in the history of Korean pharmaceutical industry. Arch Pharm Res. 2013; 36:1185–1188.

22. Kim N, Patrick L, Mair S, Stevens L, Ford G, Birks V, Lee SH. Absorption, metabolism and excretion of [14C]gemigliptin, a novel dipeptidyl peptidase 4 inhibitor, in humans. Xenobiotica. 2014; 44:522–530.

23. Hwang HJ, Chung HS, Jung TW, Ryu JY, Hong HC, Seo JA, Kim SG, Kim NH, Choi KM, Choi DS, Baik SH, Yoo HJ. The dipeptidyl peptidase-IV inhibitor inhibits the expression of vascular adhesion molecules and inflammatory cytokines in HUVECs via Akt- and AMPK-dependent mechanisms. Mol Cell Endocrinol. 2015; 405:25–34.

24. Jung GS, Kim MK, Jung YA, Kim HS, Park IS, Min BH, Lee KU, Kim JG, Park KG, Lee IK. Clusterin attenuates the development of renal fibrosis. J Am Soc Nephrol. 2012; 23:73–85.

25. Ziyadeh FN. Mediators of diabetic renal disease: the case for tgf-Beta as the major mediator. J Am Soc Nephrol. 2004; 15:Suppl 1. S55–S57.

26. Meng XM, Huang XR, Chung AC, Qin W, Shao X, Igarashi P, Ju W, Bottinger EP, Lan HY. Smad2 protects against TGF-beta/Smad3-mediated renal fibrosis. J Am Soc Nephrol. 2010; 21:1477–1487.

27. Leask A, Abraham DJ. TGF-beta signaling and the fibrotic response. FASEB J. 2004; 18:816–827.