Soluble Klotho and Fgf23/Fgfr4-Drp1 Mitochondrial Fission in CKD Cardiomyopathy

Background: Chronic kidney disease (CKD) is strongly associated with excess cardiovascular mortality that cannot be fully explained by loss of renal filtration alone. Uremic cardiomyopathy in CKD is characterized by left ventricular hypertrophy, interstitial fibrosis, mitochondrial dysfunction, and impaired energetic capacity, occurring in parallel with disturbances in mineral metabolism. Elevated fibroblast growth factor 23 (FGF23) and reduced Klotho levels have been implicated in the CKD–mineral bone disorder axis, with emerging evidence suggesting direct cardiac effects of FGF23 via FGFR4-mediated signaling pathways leading to mitochondrial fission through Drp1 activation. Objective: This study aimed to determine whether CKD-associated elevations in FGF23 contribute to cardiac remodeling through FGFR4-dependent signaling and whether mitochondrial fission via Drp1 acts as a downstream effector mechanism in uremic cardiomyopathy. Methods: A randomized, controlled, blinded preclinical study was conducted at the Toxicology Laboratory, University of Veterinary and Animal Sciences (UVAS), Lahore, Pakistan, from February 2024 to July 2025. The study employed a male Sprague–Dawley rat 5/6 nephrectomy (PNx) model, complemented by a human iPSC-derived cardiomyocyte system. Animals were randomized after CKD confirmation and allocated to five groups: Sham + vehicle, PNx + vehicle (CKD control), PNx + recombinant Klotho (rescue arm), PNx + FGFR4 blockade (mechanistic inhibition), and PNx + anti-FGF23 antibody (ligand neutralization). Cardiac structure and function were assessed using echocardiography at baseline, pre-randomization, and terminal stages. Renal and mineral metabolism profiles included serum creatinine, BUN, phosphate, calcium, PTH, FGF23, and soluble Klotho. Cardiac tissue was analyzed for fibrosis, hypertrophy, and mitochondrial structure using transmission electron microscopy (TEM). Mechanistic evaluation of Drp1 phosphorylation (Ser616/Ser637) and FGFR4–PLCγ–calcineurin–NFAT signaling was performed via Western blotting. Mitochondrial morphology and function were further validated in iPSC-derived cardiomyocytes exposed to FGF23 with or without pathway modulation. Results: CKD was confirmed 2-4 weeks post-5/6 nephrectomy, with significantly elevated serum creatinine (1.2-1.8 mg/dL vs. 0.5-0.7 mg/dL in sham, p<0.05), BUN (45-65 mg/dL vs. 15-20 mg/dL, p<0.01), phosphate (7.0-9.5 mg/dL vs. 4.5-5.5 mg/dL, p<0.05), PTH (200-400 pg/mL vs. 50-100 pg/mL, p<0.01), and FGF23 (800-2000 pg/mL vs. 50-150 pg/mL, p<0.001), while soluble Klotho was significantly reduced (100-200 pg/mL vs. 300-500 pg/mL, p<0.01). Echocardiography demonstrated progressive LV hypertrophy and dysfunction, with fractional shortening decreasing from 45-55% at baseline to 25-35% (p<0.001) by week 7-10. TEM analysis revealed significant mitochondrial fragmentation in CKD hearts, with mitochondrial length reduced from 2.5-3.5 μm to 1.0-1.8 μm (p<0.001) and mitochondrial damage increasing from <5% to 35-55% (p<0.001). Western blot analysis showed marked activation of Drp1 Ser616 phosphorylation (p-Drp1 Ser616/Total Drp1: 1.2-1.8 vs. 0.2-0.4 in sham, p<0.001) and downregulation of fusion proteins MFN1, MFN2, and OPA1. Recombinant Klotho treatment and FGFR4 blockade significantly attenuated these mitochondrial abnormalities, with mitochondrial length recovering to 2.0-2.8 μm (p<0.01) and p-Drp1 Ser616 decreasing to 0.5-0.8 (p<0.01), despite persistent renal dysfunction. FGFR4 blockade and rKlotho treatment preserved LV function (ejection fraction: 65-70% vs. 45-55% in CKD, p<0.01) and reduced fibrosis. Human iPSC-derived cardiomyocytes exposed to FGF23 (100 ng/mL) recapitulated the mitochondrial fission phenotype, with reduced mitochondrial length (0.8-1.5 μm vs. 2.0-3.0 μm in control, p<0.001), increased p-Drp1 Ser616 (1.5-2.5 vs. 0.3-0.5, p<0.001), and decreased mitochondrial membrane potential (55-65% vs. 100%, p<0.001). Co-treatment with rKlotho or FGFR4 blockade prevented these effects. Anti-FGF23 neutralization showed beneficial cardiac effects but was associated with hyperphosphatemia and increased vascular calcification risk. Conclusion: This integrated multi-system approach demonstrates that CKD-associated elevations in FGF23 contribute to cardiac remodeling through FGFR4-dependent signaling, with mitochondrial fission via Drp1 Ser616 phosphorylation acting as a critical downstream effector mechanism in uremic cardiomyopathy. Recombinant soluble Klotho attenuates cardiac mitochondrial injury and Drp1 activation even when renal parenchymal injury persists, suggesting a primarily cardioprotective role independent of renal recovery. These findings position the FGF23–Klotho–FGFR4–Drp1 axis as a potential therapeutic target for mitigating cardiovascular complications in CKD.