The Role of the Klotho KL1 Domain in CKD-Associated Cardiac Fibrosis

Abstract

Cardiac fibrosis is a major contributor to cardiovascular morbidity and mortality in patients with advanced Chronic Kidney Disease (CKD). Cardiac fibrosis is associated with multiple adverse consequences, such as impaired cardiovascular functional capacity, diastolic dysfunction, arrhythmias, and sudden cardiac death. There are currently no clinically available direct therapies for cardiac fibrosis. Klotho is a pleiotropic protein that exhibits powerful cardioprotective effects. Klotho knockout models develop severe cardiac fibrosis, conversely Klotho replacement ameliorates this condition. Klotho can inhibit several pro-fibrotic pathways that are organized by lipid rafts, and this is independent of the phosphatonin, Fibroblast Growth Factor 23 (FGF23), which is elevated in CKD. KL1 is a fragment of Klotho that does not bind with FGF23 and may exert Klotho’s FGF23-independent cardioprotective effects with fewer off-target effects than Klotho. Therefore, KL1 is of significant therapeutic interest. However, to-date, no studies have investigated the role and mechanisms of KL1’s anti-fibrotic effects at the heart in CKD. The overall aim of my dissertation was to elucidate KL1’s anti-fibrotic role and mechanisms in CKD-associated cardiac fibrosis. My overarching hypothesis is that KL1 can directly inhibit CKD-associated cardiac fibrosis via the focal adhesion pathway. In Aim 1, I demonstrated that serum Klotho was associated with procollagen type 1 C-terminal peptide, a byproduct of fibrillogenesis, in a cohort of dialysis-dependent advanced CKD patients. In Aim 2, I developed a novel Klotho mass spectrometry assay to overcome limitations of current assays and simultaneously distinguish between Klotho isoforms. In Aim 3, using a progressive CKD rat model, I found that KL1 treatment reduced cardiac fibrosis and pro-fibrotic myofibroblast markers. Using human cardiac fibroblasts exposed to mineral stressors (i.e., high phosphate and calcium) found in CKD, I demonstrated that KL1 reduced collagen 1:3 ratio. In Aim 4, I found that KL1 reduced focal adhesion kinase (FAK) protein expression, in vitro, and trended to reduced cardiac FAK transcriptional expression, in vivo. I also found that KL1 colocalized to lipid rafts of human cardiac fibroblasts, in vitro. In summary, these findings suggest that KL1 can inhibit cardiac fibrosis in CKD via the focal adhesion pathway.