Finerenone attenuates downregulation of the kidney GLP-1 receptor and glucagon receptor and cardiac GIP receptor in mice with comorbid diabetes - PubMed (original) (raw)
Finerenone attenuates downregulation of the kidney GLP-1 receptor and glucagon receptor and cardiac GIP receptor in mice with comorbid diabetes
Duc Tin Tran et al. Diabetol Metab Syndr. 2024.
Abstract
Background: Several new treatments have recently been shown to have heart and kidney protective benefits in people with diabetes. Because these treatments were developed in parallel, it is unclear how the different molecular pathways affected by the therapies may overlap. Here, we examined the effects of the mineralocorticoid receptor antagonist finerenone in mice with comorbid diabetes, focusing on the regulation of expression of the glucagon-like peptide-1 receptor (GLP-1R), gastric inhibitory polypeptide receptor (GIPR) and glucagon receptor (GCGR), which are targets of approved or investigational therapies in diabetes.
Methods: Male C57BL/6J mice were fed a high fat diet for 26 weeks. Twelve weeks into the high fat diet feeding period, mice received an intraperitoneal injection of streptozotocin before being followed for the remaining 14 weeks (DMHFD mice). After 26 weeks, mice were fed a high fat diet containing finerenone (100 mg/kg diet) or high fat diet alone for a further 2 weeks. Cell culture experiments were performed in primary vascular smooth muscle cells (VSMCs), NRK-49 F fibroblasts, HK-2 cells, and MDCK cells.
Results: DMHFD mice developed albuminuria, glomerular mesangial expansion, and diastolic dysfunction (decreased E/A ratio). Glp1r and Gcgr were predominantly expressed in arteriolar VSMCs and distal nephron structures of mouse kidneys respectively, whereas Gipr was the predominant of the three transcripts in mouse hearts. Kidney Glp1r and Gcgr and cardiac Gipr mRNA levels were reduced in DMHFD mice and this reduction was negated or attenuated with finerenone. Mechanistically, finerenone attenuated upregulation of the profibrotic growth factor Ccn2 in DMHFD kidneys, whereas recombinant CCN2 downregulated Glp1r and Gcgr in VSMCs and MDCK cells respectively.
Conclusions: Through its anti-fibrotic actions, finerenone reverses Glp1r and Gcgr downregulation in the diabetic kidney. Both finerenone and GLP-1R agonists have proven cardiorenal benefits, whereas receptor co-agonists are approved or under development. The current findings provide preclinical rationale for the combined use of finerenone with the GLP-1R agonist family. They also provide mechanism of action insights into the potential benefit of finerenone in people with diabetes for whom GLP-1R agonists or co-agonists may not be indicated.
Keywords: CCN2; CTGF; Diabetic nephropathy; Finerenone; GLP-1; GLP-1 receptor; Gastric inhibitory polypeptide; Glucagon; Glucagon receptor; Mineralocorticoid receptor.
© 2024. The Author(s).
Conflict of interest statement
Declarations. Ethical approval and consent to participate: All experimental procedures adhered to the guidelines of the Canadian Council of Animal Care and were approved by the St. Michael’s Hospital Animal Care Committee. Consent for publication: Not applicable. Competing interests: A.A. has received research support through his institution from Boehringer Ingelheim.
Figures
Fig. 1
Kidney phenotyping of age-matched control mice (control), diabetic high fat-diet fed mice (DMHFD), or DMHFD mice treated with finerenone (100 mg/kg diet) in high fat diet for the final 2 weeks (DMHFD + Finerenone). (A) Study design. (B) Urine albumin excretion in control (n = 7), DMHFD (n = 11) and DMHFD + Finerenone (n = 11) mice. (C) Glomerular volume determined on H&E stained kidney sections in control (n = 7), DMHFD (n = 18) and DMHFD + Finerenone (n = 17) mice. (D) Representative photomicrographs of kidney periodic acid-Schiff staining and quantitation of glomerulosclerosis index in control (n = 7), DMHFD (n = 17) and DMHFD + Finerenone (n = 17) mice. Original magnification x400. Scale bar = 50 μm. (E) Representative photomicrographs of kidney picrosirius red staining and quantitation of kidney picrosirius red in control (n = 7), DMHFD (n = 17) and DMHFD + Finerenone (n = 17) mice. Original magnification x100. Scale bar = 100 μm. Values are mean ± S.D. *P < 0.05, **P < 0.01 by one-way ANOVA followed by Fisher’s least significant difference test. Skew distributed data in (B) were log transformed before statistical comparison
Fig. 2
Immune cell populations and Ifng mRNA levels in the kidneys of age-matched control mice (control), diabetic high fat-diet fed mice (DMHFD), or DMHFD mice treated with finerenone (100 mg/kg diet) in high fat diet for the final 2 weeks (DMHFD + Finerenone). (A) Flow cytometry for CD45+ cells, CD45+Ly6C+ cells, infiltrating macrophages, dendritic cells and CD4+ T cells (n = 5/group). (B) qRT-PCR for Ifng in the kidneys of control (n = 7), DMHFD (n = 11) and DMHFD + Finerenone (n = 12) mice. Values are mean ± S.D. *P < 0.05, **P < 0.01 by one-way ANOVA followed by Fisher’s least significant difference test
Fig. 3
E/A ratio and cardiac histopathology in age-matched control mice (control), diabetic high fat-diet fed mice (DMHFD), or DMHFD mice treated with finerenone (100 mg/kg diet) in high fat diet for the final 2 weeks (DMHFD + Finerenone). (A) E/A ratio before treatment (26 weeks (wks)) and two weeks after finerenone or vehicle (28 wks). Control (n = 5), DMHFD (n = 13), DMHFD + Finerenone (n = 12). (B) Representative H&E stained heart sections and cardiomyocyte cross sectional area determined on H&E-stained kidney sections in control (n = 7), DMHFD (n = 18), DMHFD + Finerenone (n = 16). Original magnification x400. Scale bar = 50 μm. (C) Representative photomicrographs of cardiac picrosirius red staining and quantitation of cardiac picrosirius red in control (n = 7), DMHFD (n = 18) and DMHFD + Finerenone (n = 16) mice. Original magnification x100. Scale bar = 100 μm. (D) Representative photomicrographs of isolectin B4 staining and quantitation of cardiac capillary density in control (n = 7), DMHFD (n = 18) and DMHFD + Finerenone (n = 17) mice. Original magnification x400. Scale bar = 50 μm. Values are mean ± S.D. **P < 0.01 by two-tailed paired Student t test (A) or by one-way ANOVA followed by Fisher’s least significant difference test (B)
Fig. 4
RNAscope in situ hybridization for Glp1r, Gipr and Gcgr in mouse kidneys and hearts. dapB = negative control. (A) Representative kidney sections and quantitation of RNAscope puncta per x400 magnification field in control mouse kidneys (n = 5/transcript). Original magnification x400. Scale bar = 50 μm. (B) Quantitation of RNAscope puncta per x400 magnification field in glomeruli, proximal tubule cells, distal nephron cells, vascular smooth muscle cells (VSMCs) and interstitial cells in mouse kidney sections (n = 5/transcript). (C) Representative cardiac cross sections and mean RNAscope puncta for Glp1r, Gipr, Gcgr and dapB per x400 magnification field in control mouse hearts (n = 5/transcript). Original magnification x400. Scale bar = 50 μm. Values are mean ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc test
Fig. 5
Finerenone negates kidney Glp1r and Gcgr and cardiac Gipr downregulation in mice with comorbid diabetes. qRT-PCR for kidney Glp1r, kidney Gcgr, cardiac Gipr, and kidney and heart Tgfb1, Tnfa and Ccn2 in age-matched control mice (control), diabetic high fat-diet fed mice (DMHFD), or DMHFD mice treated with finerenone (100 mg/kg diet) in high fat diet for the final 2 weeks (DMHFD + Finerenone). Kidney Glp1r (A), kidney Gcgr (B), heart Gipr (C), kidney Tgfb1 (D), kidney Tnfa (E), kidney Ccn2 (F), heart Tgfb1 (G), heart Tnfa (H), heart Ccn2 (I) in control (n = 7), DMHFD (n = 11), DMHFD + Finerenone (n = 12); except (A) DMHFD (n = 10), and (E) DMHFD + Finerenone (n = 10). Values are mean ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA followed by Fisher’s least significant difference test
Fig. 6
Aldosterone upregulates CCN2 in NRK-49 F cells, HK-2 cells and MDCK cells, and CCN2 downregulates Glp1r in mouse vascular smooth muscle cells (VSMCs) and Gcgr in MDCK cells. (A and B) Characterization of primary cultured VSMCs. (A) Phase-contrast microscopy image of VSMCs. Original magnification x100. Scale bar = 50 μm. (B) Immunofluorescence staining for the smooth muscle cell marker transgelin. Scale bar = 50 μm. Experiment performed in duplicate. (C-F) qRT-PCR for CCN2 in NRK-49 F cells (C), HK-2 cells (D), MDCK cells (E) and VSMCs (F) treated under control conditions, or with 100nmol/L aldosterone for 24 h (n = 6/condition). (G) qRT-PCR for Ccn2 in NRK-49 F cells treated in the presence or absence of 100nmol/L aldosterone with or without finerenone (5µmol/L, 50µmol/L, or 100µmol/L) (n = 6/condition). (H and I) qRT-PCR for Glp1r in VSMCs (H) or Gcgr in MDCK cells (I) under control conditions, or with 100ng/mL recombinant CCN2 for 24 h (n = 6/condition). Values are mean ± S.D. **P < 0.01, ***P < 0.001, ****P < 0.0001 by unpaired two-tailed Student t test (C, D, E, F, H, I) or one-way ANOVA with Sidak’s post-test (G)
References
- Urva S, Coskun T, Loh MT, Du Y, Thomas MK, Gurbuz S, et al. LY3437943, a novel triple GIP, GLP-1, and glucagon receptor agonist in people with type 2 diabetes: a phase 1b, multicentre, double-blind, placebo-controlled, randomised, multiple-ascending dose trial. Lancet. 2022;400(10366):1869–81. - DOI - PubMed
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