Fig. 1Effect of gemigliptin (Gemi) on unilateral ureteral obstruction (UUO)-induced renopathological changes. (A) Representative images of H&E and Sirius red staining of kidney tissue sections from control (CON) mice and UUO mice without or with Gemi treatment (300 mg/kg; UUO+Gemi). 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 were quantitated by computer-based morphometric analysis. All morphometric data were normalized against the corresponding values in CON animals. Data in all bar graphs are expressed as fold increase relative to the CON (n=6 in each group). (B) Representative images of immunohistochemical staining for fibronectin, plasminogen activator inhibitor 1 (PAI-1), and type I collagen in kidney tissue sections from CON mice or UUO mice without or with Gemi treatment (300 mg/kg; UUO+Gemi). Areas of positive staining with fibronectin, PAI-1, and type I collagen antibodies were quantitated by computer-based morphometric analysis. All data were expressed as the mean±standard error of the mean (SEM) of five random fields from each kidney section (n=6 in each group). (C, D) Representative Western blot analysis of renal protein levels of fibronectin, PAI-1, and type I collagen normalized to 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, dP<0.01 compared with UUO-induced mice.
Fig. 2Effect of gemigliptin (Gemi) on the unilateral ureteral obstruction (UUO)-induced inflammasome. (A) Representative images of immunohistochemical staining for NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, and interleukin 1β (IL-1β) in kidney tissue sections from control (CON) mice or UUO mice without or with Gemi treatment (300 mg/kg; UUO+Gemi). Areas of positive staining for NLRP3, ASC, caspase-1, and IL-1β were quantitated by computer-based morphometric analysis. All data were expressed as the mean±standard error of the mean (SEM) of five random fields from each kidney section (n=6 in each group). (B, C) Representative Western blot analysis of renal protein levels of NLRP3, ASC, caspase-1, and IL-1β normalized to 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, cP<0.05 compared with CON mice, dP<0.001, eP<0.01, fP<0.05 compared with UUO-induced mice.
Fig. 3Effects of gemigliptin (Gemi) on transforming growth factor-β (TGF-β)-stimulated fibrosis- and inflammasome-related gene expression in human proximal tubule epithelial cells. (A, B) Representative Western blot analyses of the level of type I collagen, α smooth muscle actin (α-SMA), and plasminogen activator inhibitor 1 (PAI-1) in TGF-β-stimulated human renal proximal tubule cells (HK-2) normalized to β-tubulin. Quantitation of Western blot analyses in TGF-β-stimulated HK-2 cells. Data are the mean±standard error of the mean (SEM) of three independent measurements (three separate experiments). (C, D) Representative Western blot analyses of the level of NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, and interleukin 1β (IL-1β) in TGF-β-stimulated HK-2 cells. Quantitation of Western blot analyses of TGF-β-stimulated HK-2 cells. aP<0.001, bP<0.01, cP<0.05 compared with control, dP<0.001, eP<0.01, fP<0.05 compared with TGF-β alone.
Fig. 4Effects of gemigliptin (Gemi) on nuclear factor-κB (NF-κB) activation in vivo and in vitro. (A) Representative images of immunohistochemical staining for phosphorylated NF-κB (p-NF-κB) and CD-26 in kidney tissue sections from control (CON) mice or unilateral ureteral obstruction (UUO) mice without or with Gemi treatment (300 mg/kg; UUO+Gemi). Areas of positive staining with p-NF-κB antibody were quantitated by computer-based morphometric analysis. All data were expressed as the mean±standard error of the mean (SEM) of five random fields from each kidney section (n=6 in each group). (B, C) Representative Western blot analysis of levels of p-NF-κB normalized to β-tubulin. The data are represented as the mean±SEM of three independent measurements (n=6 in each group). (D, E) Representative Western blot analyses of the level of p-NF-κB in transforming growth factor-β (TGF-β)-stimulated human renal proximal tubule cells (HK-2) normalized to β-tubulin. Quantitation of Western blot analyses in TGF-β-stimulated HK-2 cells. Data are the mean±SEM of three independent measurements (three separate experiments). aP<0.001 compared with CON mice, bP<0.001 compared with UUO-induced mice, cP<0.001 compared with control, dP<0.01 compared with TGF-β alone.