The kisspeptin-1 receptor antagonist peptide-234 aggravates uremic cardiomyopathy in a rat model

Abstract

Uremic cardiomyopathy is characterized by diastolic dysfunction, left ventricular hypertrophy (LVH), and fibrosis. Dysregulation of the kisspeptin receptor (KISS1R)-mediated pathways are associated with the development of fibrosis in cancerous diseases. Here, we investigated the effects of the KISS1R antagonist peptide-234 (P234) on the development of uremic cardiomyopathy. Male Wistar rats (300-350 g) were randomized into four groups: (i) Sham, (ii) chronic kidney disease (CKD) induced by 5/6 nephrectomy, (iii) CKD treated with a lower dose of P234 (ip. 13 µg/day), (iv) CKD treated with a higher dose of P234 (ip. 26 µg/day). Treatments were administered daily from week 3 for 10 days. At week 13, the P234 administration did not influence the creatinine clearance and urinary protein excretion. However, the higher dose of P234 led to reduced anterior and posterior wall thicknesses, more severe interstitial fibrosis, and overexpression of genes associated with left ventricular remodeling (Ctgf, Tgfb, Col3a1, Mmp9), stretch (Nppa), and apoptosis (Bax, Bcl2, Casp7) compared to the CKD group. In contrast, no significant differences were found in the expressions of apoptosis-associated proteins between the groups. Our results suggest that the higher dose of P234 hastens the development and pathophysiology of uremic cardiomyopathy by activating the fibrotic TGF-β-mediated pathways.

Figure 1

Experiment setup. Blood: blood sampling, BP: blood pressure, Urine: urine sampling, Echo: echocardiography, CKD: chronic kidney disease, PBS: phosphate-buffered saline, P234: KISS1R antagonist peptide-234, LV: left ventricle, Op: operation. Sham: sham-operated group, CKD: chronic kidney disease group, CKD + P234 D1: chronic kidney disease group treated with the lower dose (13 μg/day, dose 1) of KISS1R antagonist peptide-234, CKD + P234 D2: chronic kidney disease group treated with the higher dose (26 μg/day, dose 2) of KISS1R antagonist peptide-234.

Figure 2

The effects of the KISS1R antagonist peptide-234 on the development of CKD in 5/6 nephrectomized rats. (A) Serum urea concentration, (B) serum creatinine concentration, (C) creatinine clearance, (D) 24-h urinary protein excretion, (E) 24-h urinary creatinine excretion, and (F) 24-h urine volume at the endpoint. Values are presented as mean ± S.E.M., *p < 0.05 vs. sham group (n = 7–8, One-Way ANOVA, Holm-Sidak post hoc test), ^$p < 0.05 vs. the week 5 values in the same group (n = 7–8, Two-Way Repeated Measures ANOVA, Holm-Sidak post hoc test). Creatinine clearance was calculated according to the standard formula (urine creatinine concentration [μM] × urine volume for 24 h [mL])/(serum creatinine concentration [μM] × 24 × 60 min). ^¥At the endpoint, urine volume and creatinine concentration were measured at week 12 and serum creatinine concentration at week 13. Sham: sham-operated group, CKD: chronic kidney disease group, CKD + P234 D1: chronic kidney disease group treated with the lower dose (13 μg/day, dose 1) of KISS1R antagonist peptide-234, CKD + P234 D2: chronic kidney disease group treated with the higher dose (26 μg/day, dose 2) of KISS1R antagonist peptide-234.

Figure 3

The effects of the KISS1R antagonist peptide-234 on the echocardiographic parameters. (a) Representative M-mode images, (b) systolic posterior wall thickness (PWTs), (c) diastolic posterior wall thickness (PWTd), (d) Representative pulse wave and tissue Doppler images of mitral valve early flow velocity (e) and septal mitral annulus (e′) velocity, E) E/e′ ratio, (f) ejection fraction (EF). Values are presented as mean ± S.E.M., *p < 0.05 vs. sham, ^#p < 0.05 vs. CKD (n = 7–9, One-Way ANOVA, Holm-Sidak post hoc test). ^$p < 0.05 vs. week 5 values in the same group (n = 7–8, Two-Way Repeated-Measures ANOVA, Holm-Sidak post hoc test). Sham: sham-operated group, CKD: chronic kidney disease group, CKD + P234 D1: chronic kidney disease group treated with the lower dose (13 μg/day, dose 1) of KISS1R antagonist peptide-234, CKD + P234 D2: chronic kidney disease group treated with the higher dose (26 μg/day, dose 2) of KISS1R antagonist peptide-234.

Figure 4

The effects of the KISS1R antagonist peptide-234 on cardiomyocyte hypertrophy and interstitial fibrosis and molecular markers of heart failure at week 13. (a) Representative hematoxylin–eosin (HE)-stained slides at 100 × and 40 × magnifications and representative picrosirius red and fast green (PSFG)-stained slides at 20 × magnifcation (b) cardiomyocyte cross-sectional areas, (c) left ventricular collagen content, (d) A-type natriuretic peptide (Nppa), and (E) matrix metalloproteinase-9 (Mmp9) expressions in the left ventricles normalized to the ribosomal protein lateral stalk subunit P2 (Rplp2) gene expression. On the digital HE images, cardiomyocyte cross-sectional areas were measured in 100 selected cardiomyocytes on left ventricular sections cut on the same plane. The mean values of the collagen content of 10 representative PSFG-stained images were calculated and used for statistical evaluation in the case of each left ventricular slide. Scale bars represent 10 µm at the 100 × magnifed images, 20 µm at the 40 × magnifed images, and 50 µm at the 20 × magnifed images. Values are presented as mean ± S.E.M., *p < 0.05 vs. sham, ^#p < 0.05 vs. CKD (n = 7–8, one-way ANOVA, Holm-Sidak post hoc test). Sham: sham-operated group, CKD: chronic kidney disease group, CKD + P234 D1: chronic kidney disease group treated with the lower dose (13 μg/day, dose 1) of KISS1R antagonist peptide-234, CKD + P234 D2: chronic kidney disease group treated with the higher dose (26 μg/day, dose 2) of KISS1R antagonist peptide-234.

Figure 5

The effects of the KISS1R antagonist peptide-234 on the left ventricular expression of genes associated with inflammation and fibrosis. Relative gene expression of (a) interleukin-1 (Il1), (b), interleukin-6 (Il6), (c) tumor necrosis factor-α (Tnf), (d) connective tissue growth factor (Ctgf), (e) transforming growth factor-β (Tgfb), (f) collagen type 1 alpha 1 chain (Col1a1) and (g), collagen type 3 alpha 1 chain (Col3a1) normalized to the ribosomal protein lateral stalk subunit P2 (Rplp2) gene expression. Values are presented as mean ± S.E.M., *p < 0.05 vs. sham, ^#p < 0.05 vs. CKD (n = 7–8, One-Way ANOVA, Holm-Sidak post hoc test). Sham: sham-operated group, CKD: chronic kidney disease group, CKD + P234 D1: chronic kidney disease group treated with the lower dose (13 μg/day, dose 1) of KISS1R antagonist peptide-234, CKD + P234 D2: chronic kidney disease group treated with the higher dose (26 μg/day, dose 2) of KISS1R antagonist peptide-234.

Figure 6

The effects of the KISS1R antagonist peptide-234 on the protein levels of KISS1R and ERK1/2 at week 13. Left ventricular protein levels and cropped representative Western blot imagines of (a) Kisspeptin receptor-1 (KISS1R, 40–140 kDa), (b) total ERK1 (44 kDa), (c) phospho-ERK1 (pERK1, 44 kDa), (d) pERK1/ERK1 ratio and (e) total ERK 2 (42 kDa), (f) phospho-ERK2 (pERK2, 42 kDa), (g) pERK2/ERK2 ratio. Values are presented as mean ± S.E.M., *p < 0.05 vs. sham (n = 7, One-Way ANOVA, Holm-Sidak post hoc test). Sham: sham-operated group, CKD: chronic kidney disease group, CKD + P234 D1: chronic kidney disease group treated with the lower dose (13 μg/day, dose 1) of KISS1R antagonist peptide-234, CKD + P234 D2: chronic kidney disease group treated with the higher dose (26 μg/day, dose 2) of KISS1R antagonist peptide-234. Images were captured with the Odyssey CLx machine and exported with Image Studio 5.2.5 software. The full-length Ponceau-stained membranes and the corresponding Western blot images are presented in the Supplementary Material (Figs. S3–S5).

Figure 7

The effects of the KISS1R antagonist peptide-234 on apoptosis-associated gene expressions and protein levels in the left ventricles at week 13. Relative gene expression of (a) BCL2-associated X apoptosis regulator (Bax), (b) B-Cell CLL/lymphoma 2 apoptosis regulator (Bcl2), (c) Bax/Bcl2 ratio, and (d) caspase7 (Casp7) normalized to the ribosomal protein lateral stalk subunit P2 (Rplp2) gene expression. Left ventricular protein levels and cropped representative imagines of (e) BAX (20 kDa), (f) BCL2 (26 kDa), (g) BAX/BCL2 ratio, and (h) CASP 7 (35 kDa). Values are presented as mean ± S.E.M., *p < 0.05 vs. sham, ^#p < 0.05 vs. CKD vehicle group (n = 7–8 for RT-qPCR and n = 7 for Western blot measurements, One-Way ANOVA, Holm-Sidak post hoc test). Sham: sham-operated group, CKD: chronic kidney disease group, CKD + P234 D1: chronic kidney disease group treated with the lower dose (13 μg/day, dose 1) of KISS1R antagonist peptide-234, CKD + P234 D2: chronic kidney disease group treated with the higher dose (26 μg/day, dose 2) of KISS1R antagonist peptide-234. Images were captured with the Odyssey CLx machine and exported with Image Studio 5.2.5 software. The full-length Ponceau-stained membranes and the corresponding Western blot images are presented in the Supplementary Material (Figs. S6–S8).