Glucagon-Like Peptide-1 Mediates the Protective Effect of the Dipeptidyl Peptidase IV Inhibitor on Renal Fibrosis via Reducing the Phenotypic Conversion of Renal Microvascular Cells in Monocrotaline-Treated Rats

Abstract

Chronic kidney diseases are characterized by renal fibrosis with excessive matrix deposition, leading to a progressive loss of functional renal parenchyma and, eventually, renal failure. Renal microcirculation lesions, including the phenotypic conversion of vascular cells, contribute to renal fibrosis. Here, renal microcirculation lesions were established with monocrotaline (MCT, 60 mg/kg). Sitagliptin (40 mg/kg/d), a classical dipeptidyl peptidase-4 (DPP-4) inhibitor, attenuated the renal microcirculation lesions by inhibiting glomerular tuft hypertrophy, glomerular mesangial expansion, and microvascular thrombosis. These effects of sitagliptin were mediated by glucagon-like peptide-1 receptor (GLP-1R), since they were blocked by the GLP-1R antagonist exendin-3 (Ex-3, 40 ug/kg/d). The GLP-1R agonist liraglutide showed a similar renal protective effect in a dose-independent manner. In addition, sitagliptin, as well as liraglutide, alleviated the MCT-induced apoptosis of renal cells by increasing the expression of survival factor glucose-regulated protein 78 (GRP78), which was abolished by the GLP-1R antagonist Ex-3. Sitagliptin and liraglutide also effectively ameliorated the conversion of vascular smooth muscle cells (SMCs) from a synthetic phenotype to contractile phenotype. Moreover, sitagliptin and liraglutide inhibited endothelial-mesenchymal transition (EndMT) via downregulating transforming growth factor-β1 (TGF-β1). Collectively, these findings suggest that DPP-4 inhibition can reduce microcirculation lesion-induced renal fibrosis in a GLP-1-dependent manner.

Figure 1

Representative renal immunohistochemical staining for DPP-4.

Figure 2

Effects of sitagliptin (SG), exendin-3 (Ex-3), and liraglutide (Li) on the renal glomerulus structure and protein expression of dipeptidyl peptidase-4 (DPP-4) and glucagon-like peptide-1 (GLP-1) in the kidney during monocrotaline- (MCT-) induced renal injury. (a) Representative renal histological staining with haematoxylin and eosin (HE) and periodic acid-Schiff (PAS). (b) Graphic analysis of the average glomerulus surface area according to the HE staining. (c) Graphic analysis of the degree of glomerular mesangial expansion according to the PAS staining. (d) Representative western blots of DPP-4 and GLP-1. (e, f) Immunoblot analysis of DPP-4 and GLP-1. Data are expressed as the means ± SD; n = 6–8 rats in each group; ^#P < 0.05 versus control (Con); ^∗P < 0.05 versus MCT; ^§P < 0.05 versus MCT + 40 mg/kg SG.

Figure 3

Effects of sitagliptin, exendin-3, and liraglutide on MCT-induced renal cells apoptosis. (a) Representative renal histological staining with HE, immunohistochemical staining for caspase 3 and proliferating cell nuclear antigen (PCNA), and TUNEL staining. (b) Graphic analysis of immunohistochemistry for caspase 3. (c) Graphic analysis of TUNEL (×200). (d) Graphic analysis of immunohistochemistry for PCNA (×200). (e, f, g, h) Representative western blots and immunoblot analysis of cleaved-caspase 3 (c-caspase 3), glucose-regulated protein 78 (GRP78), Bcl2, and Bax. Data are expressed as the means ± SD; n = 6–8 rats in each group; ^#P < 0.05 versus control (Con); ^∗P < 0.05 versus MCT; ^§P < 0.05 versus MCT + 40 mg/kg SG.

Figure 4

Effects of sitagliptin, exendin-3, and liraglutide on MCT-induced renal fibrosis and the phenotypic conversion of smooth muscle cells. (a) Histological staining with the Masson trichrome stain (MTS) and immunohistochemical staining for CD68 and α-smooth muscle actin (αSMA). (b) Graphic analysis of MTS. (c) Graphic analysis of immunohistochemistry for CD68 (×400). (d) Graphic analysis of αSMA thickness. (e, f) Representative western blots and immunoblot analysis of smooth muscle 22 alpha (SM22α). Data are expressed as the means ± SD; n = 6–8 rats in each group; ^#P < 0.05 versus control (Con); ^∗P < 0.05 versus MCT; ^§P < 0.05 versus MCT + 40 mg/kg SG.

Figure 5

Effects of sitagliptin, exendin 3, and liraglutide on MCT-induced endothelial-to-mesenchymal transition (EndMT). (a) Representative immunohistochemical staining for CD31 (Red), αSMA (Green) and CD31 (Green), SM22α (Red). (b) Representative western blots and immunoblot analysis of von Willebrand factor (vWF), αSMA, transforming growth factor-β1 (TGF-β1), TGFβ receptor 1 (TGFβR1), phosphorylated Smad3/Smad 3, bone morphogenetic protein receptor type 2 (BMPR2), and Snail. Data are expressed as the means ± SD; n = 6–8 rats in each group; ^#P < 0.05 versus control (Con); ^∗P < 0.05 versus MCT; ^§P < 0.05 versus MCT + 40 mg/kg SG.

Figure 6

Dipeptidyl peptidase-4 (DPP-4) inhibition with sitagliptin decreases the degranulation of glucagon-like peptide-1 (GLP-1) 7-36 (active form) and promotes the transduction of GLP-1R signalling, which can be blocked by the GLP-1R antagonist exendin-3. The GLP-1R agonist liraglutide can activate GLP-1R signalling directly. In smooth muscle cells (SMCs), the activation of GLP-1R upregulates the expression of smooth muscle 22 alpha (SM22α), which is expressed at high levels in the contractile phenotype of SMCs, and inhibits the transition of SMCs from the contractile phenotype to the synthetic phenotype. The extracellular matrix derived from the synthetic phenotype of SMCs is then obviously reduced. In endothelial cells, activated GLP-1R signalling can upregulate bone morphogenetic protein receptor type 2 (BMPR2) expression and reduce transforming growth factor-β1 (TGF-β1)/Smad3 signalling, followed by inhibiting Snail expression. Then, the protein expression of endothelial marker von Willebrand factor (vWF) increases, while that of the mesenchymal marker α-smooth muscle actin (αSMA) decreases. After that, the endothelial-mesenchymal transition (EndMT) programme is blocked, the extracellular matrix derived from myofibroblasts is reduced, and, ultimately, renal fibrosis is attenuated.