Fig. 1Effect 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. 2Effect 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. 3Effect 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. 4Effects 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. 5Effects 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.