Diabetes Metab J.  2019 Dec;43(6):830-839. 10.4093/dmj.2018.0181.

Gemigliptin Attenuates Renal Fibrosis Through Down-Regulation of the NLRP3 Inflammasome

• Seo JB
• Choi YK
• Woo HI
• Jung YA
• Lee S
• Lee S
• Park M
• Lee IK
• Jung GS
• Park KG

• KMID: 2466504
• DOI: http://doi.org/10.4093/dmj.2018.0181

Abstract

BACKGROUND
The hypoglycemic drugs dipeptidyl peptidase-4 (DPP-4) inhibitors have proven protective effects on diabetic kidney disease, including renal fibrosis. Although NOD-like receptor protein 3 (NLRP3) inflammasome activation is known to play an important role in the progression of renal fibrosis, the impact of DPP-4 inhibition on NLRP3-mediated inflammation while ameliorating renal fibrosis has not been fully elucidated. Here, we report that the renoprotective effect of gemigliptin is associated with a reduction in NLRP3-mediated inflammation in a murine model of renal fibrosis.
METHODS
We examined the effects of gemigliptin on renal tubulointerstitial fibrosis induced in mice by unilateral ureteral obstruction (UUO). Using immunohistochemical and Western blot analysis, we quantitated components of the NLRP3 inflammasome in kidneys with and without gemigliptin treatment, and in vitro in human kidney tubular epithelial human renal proximal tubule cells (HK-2) cells, we further analyzed the effect of gemigliptin on transforming growth factor-β (TGF-β)-stimulated production of profibrotic proteins.
RESULTS
Immunohistological examination revealed that gemigliptin ameliorated UUO-induced tubular atrophy and renal fibrosis. Gemigliptin-treated kidneys showed a reduction in levels of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, and interleukin-1β, which had all been markedly increased by UUO. In line with the in vivo
results
, TGF-β markedly increased NLRP3 inflammasome markers, which were attenuated by gemigliptin treatment. Furthermore, gemigliptin treatment attenuated phosphorylated nuclear factor-κB levels, which had been increased in the UUO kidney as well as in TGF-β-treated cultured renal cells.
CONCLUSION
The present study shows that activation of the NLRP3 inflammasome contributes to UUO-induced renal fibrosis and the renoprotective effect of gemigliptin is associated with attenuation of NLRP3 inflammasome activation.

Keyword

Dipeptidyl-peptidase IV inhibitors; Fibrosis; Inflammasomes; Kidney; LC15-0444 (Gemigliptin)

MeSH Terms

Animals
Atrophy
Blotting, Western
Diabetic Nephropathies
Dipeptidyl-Peptidase IV Inhibitors
Down-Regulation*
Fibrosis*
Humans
Hypoglycemic Agents
In Vitro Techniques
Inflammasomes*
Inflammation
Kidney
Mice
Ureteral Obstruction
Dipeptidyl-Peptidase IV Inhibitors
Hypoglycemic Agents
Inflammasomes

Figure

• 

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

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