DPP-4 inhibition enhanced renal tubular and myocardial GLP-1 receptor expression decreased in CKD with myocardial infarction

Figures

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  Fig. 1. Experimental design. CKD, 5/6 nephrectomy; MI/R, myocardial ischemia reperfusion; CKD + MI/R, 5/6 nephrectomy with myocardial ischemia reperfusion. (n = 6 each). a Experimental design I b Experimental design II c Experimental design III

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  Fig. 2. Kaplan–Meier plot for conditions of control, CKD, CKD-Li, CKD-MI/R and CKD-MI/R-Li associated with animal survival. CKD, chronic kidney disease; MI/R, myocardial ischemia/reperfusion; Li, linagliptin treatment

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  Fig. 3. Dipeptidyl-peptidase 4 (DPP-4) activity in chronic kidney disease (CKD) and CKD with linagliptin-treated rats. Control, sham-operated; CKD 2, 4, 8 weeks, 5/6 nephrectomized rats 2, 4, and 8 weeks post-treatment; Lina, linagliptin. Values are presented as the mean ± standard deviation (SD)

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  Fig. 4. Change in treatment outcomes in CKD and CKD with linagliptin-treated rats. Blood pressure (a), urine volume (b), blood urea nitrogen (c), serum creatinine (d), albumin (e), and creatinine clearance (f). Control, sham-operated; CKD 2, 4, and 8 weeks, 5/6 nephrectomized rats 2, 4, and 8 weeks post-treatment; Lina, linagliptin. Values are presented as the mean ± SD. *p < 0.05 vs. control; **p < 0.01 vs. control; †p < 0.05 vs. CKD, 2 weeks; ††p < 0.01 vs. CKD, 2 weeks; ‡p < 0.05 vs. CKD, 4 weeks; ‡‡p < 0.01 vs. CKD, 4 weeks; ¥p < 0.05 vs. CKD, 8 weeks; and ¥¥p < 0.01 vs. CKD, 8 weeks

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  Fig. 5. Glucagon-like peptide-1 receptor (GLP-1R) expression in the CKD rat. a Western blot analysis of GLP-1R expression in rat kidney proximal tubule (NRK-52E) cells with hypoxic injury. b Western blot analysis of renal cortical GLP-1R expression in 5/6 nephrectomized rat. c Renal cortical GLP-1R immunoreactivity in CKD rats. Control, sham-operated; CKD 2, 4, 8 weeks, 5/6 nephrectomized rats 2, 4, and 8 weeks post-treatment. Black lines, separated from adjacent bands. Values are presented as the mean ± SD. *p < 0.05 vs. control. Magnification 400 ×. * The original blots of fig 5-11 are in Additional file 1: Figure S1.

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  Fig. 6. Effect of linagliptin in CKD and CKD with linagliptin-treated rats. A Western blot analysis of CKD and CKD with linagliptin-treated rats. B GLP-1R immunoreactivity in the renal cortical proximal tubule of CKD and CKD with linagliptin-treated rats. a, control; b, CKD, 2 wks; c, CKD, 2 weeks with linagliptin-treated; d, CKD, 4 wks; e, CKD, 4 weeks with linagliptin-treated; f, CKD, 8 wks; g, CKD, 8 weeks with linagliptin-treated; Values are presented as the mean ± SD. *p < 0.05 vs. control; **p < 0.01 vs. control; †p < 0.05 vs. CKD, 2 weeks; ††p < 0.01 vs. CKD, 2 weeks; ‡p < 0.05 vs. CKD, 4 weeks; ‡‡p < 0.01 vs. CKD, 4 weeks; ¥p < 0.05 vs. CKD 8, weeks; and ¥¥p < 0.01 vs. CKD, 8 weeks. Magnification 400 ×

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  Fig. 7. Expression of renal tubular GLP-1R. A Western blots showing GLP-1R protein levels in the renal cortex of control; CKD, 8 weeks; CKD, 8 weeks + Lina; CKD, 8 weeks + MI/R; and CKD, 8 weeks + MI/R + Lina rats. B Quantification of GLP-1R protein levels were standardized based on β-actin expression in the renal cortex. C GLP-1R immunoreactivity of the renal cortex. a, f, and k, control; b, g, and i, CKD, 8 weeks; c, h, and m, CKD, 8 weeks with linagliptin-treated; d, i, and n, CKD, 8 weeks and MI/R; e, j, and o, CKD, 8 weeks and MI/R with linagliptin-treated group. CKD, chronic kidney disease; MI/R, myocardial ischemia/reperfusion; Lina, linagliptin. Black lines, separated from adjacent bands. Values are presented as the mean ± SD. *p < 0.05 vs. control. a–e, magnification 100×; f–o, magnification 400 ×

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  Fig. 8. GLP-1R in cardiac progenitor cells and myocardium by ischemia. A Western blot of cardiac progenitor cells with hypoxic injury. B GLP-1R Immunoreactivity of myocardium. a and e, control; b and f, MI/R 1 day; c and g, MI/R 1 days; d and h, MI/R 7 days; i and l, MI/R 1 day with linagliptin-treated; j and m, MI/R 3 day with linagliptin-treated; k and n, MI/R 7 day with linagliptin-treated group. C Masson’s Trichrome staning of myocardium. a, control; b, MI/R 1 day; c, MI/R 1 day with linagliptin-treated; d, MI/R 3 days; e, MI/R 3 day with linagliptin-treated; f, MI/R 7 day; g, MI/R 7 day with linagliptin-treated group. MI/R, myocardial ischemia/reperfusion; Lina, linagliptin. A a-d and i-k, magnification 200×; e-h and l-m, magnification 400×. C a-g, magnification 40 ×

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  Fig. 9. Expression of myocardial GLP-1R. A Western blot analysis of protein levels in the myocardium of control; CKD, 8 weeks; CKD, 8 weeks + Lina; CKD, 8 weeks + MI/R; and CKD, 8 weeks + MI/R + Lina rats. B Quantification of GLP-1R protein levels was standardized based on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression in the myocardium. C GLP-1R immunoreactivity of myocardium. a and f, control; b and g, CKD, 8 weeks; c and h, CKD, 8 weeks with linagliptin-treated; d and i, CKD, 8 weeks and MI/R; e and j, CKD, 8 weeks and MI/R with linagliptin-treated group. D Masson’s Trichrome staning of myocardium. a, control; b, CKD, 8 weeks; c, CKD, 8 weeks with linagliptin-treated; d, CKD, 8 weeks and MI/R; e, CKD, 8 weeks and MI/R with linagliptin-treated group. CKD, chronic kidney disease; MI/R, myocardial ischemia/reperfusion; Lina, linagliptin. Values are presented as the mean ± SD. *p < 0.05 vs. control. B a–e, magnification 100×; f–o, magnification 400×, C a–e, magnification 100 ×

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  Fig. 10. Phosphorylation of extracellular signal-regulated kinase (EKR1/2) and expression of B cell lymphoma-2 (Bcl-2) in the renal cortex. a Western blot showing protein levels in renal cortex from control; CKD, 8 weeks; CKD, 8 weeks + Lina; CKD, 8 weeks + MI/R; CKD, 8 weeks + MI/R + Lina. b and c Quantification of p-ERK1/2 and Bcl-2 protein levels was standardized relative to t-ERK1/2, and β-actin expression in the renal cortex. Values are presented as the mean ± SD. *p < 0.05 vs. control; **p < 0.01 vs. control; #p < 0.05 vs. CKD, 8 weeks; †p < 0.05 vs. CKD, 8 weeks + MI/R

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  Fig. 11. Phosphorylation of EKR1/2 and expression of Bcl-2 in the myocardium. a Western blot showing protein levels in control, CKD with/without linagliptin, and CKD-MI with/without linagliptin rats. b and c Quantitation of p-ERK1/2 and Bcl-2 standardized relative to t-ERK1/2, and GAPDH expression in the renal cortex. *p < 0.05 vs. control; #p < 0.05 vs. CKD, 8 weeks; †p < 0.05 vs. CKD, 8 weeks + MI/R; ††p < 0.01 vs. CKD, 8 weeks + MI/R