Background: We previously identified raf kinase inhibitor protein (RKIP) as a relevant genetic risk factor for left ventricular (LV) remodeling: systemic RKIP-deficiency reduces pressure-overload induced cardiac fibrosis as well as oxidative stress in C57BL/6N mice. Chronic kidney disease (CKD) leads to cardiac remodeling and eventually heart failure. Here, we tested the hypothesis whether RKIP is a regulator of uremia-induced myocardial fibrosis.

Methods and Results: 10-week-old male wild-type (WT) and systemic RKIP-deficient (RKIP-/-) C57BL/6N (N) mice underwent 5/6 nephrectomy (5/6 Nx, remnant kidney model, RKM) or sham operation for 10 weeks. Following RKM, both WT and RKIP-/- mice demonstrated significantly enhanced plasma creatinine and urea levels. The peripheral blood pressure measured by the tail-cuff method was not significantly changed after 9 weeks post 5/6 Nx in both groups. In RKIP-/- mice histological analyses revealed reduction of RKM-induced LV fibrosis (picrosirius red staining) (WT SHAM 4±0.4%, RKIP-/- SHAM 4±0.5%; WT RKM 9.5±1.3%, vs. RKIP-/- RKM 5±0.9%, p=0.002) and cardiomyocyte hypertrophy (WT SHAM 274±30, RKIP-/- SHAM 259±15/µm²; WT RKM 384±36, vs. RKIP-/- RKM 304±17/µm², p=0.04). Likewise, RKIP-deficiency diminished the number of LV fibroblasts/mm² in the RKM group (WT SHAM 3543±435, RKIP-/- SHAM 3456±360; WT RKM 6580±606, vs. RKIP-/- RKM 3553±505, p=1*10^-4) and LV expression of collagen Iα2 mRNA in CKD. After RKM, systemic RKIP-deficiency reduced oxidative stress in cardiac fibroblasts measured by the co-immunostaining for intracellular fibronectin and 8-hydroxyguanosine (WT SHAM 64±3%, RKIP-/- SHAM 68±5%; WT RKM 85±2%, vs. RKIP-/- RKM 63±3%, p=3*10^-4). The percentage of CXCR4⁺ fibroblasts in the LV myocardium was increased by RKM (WT RKM 75±4%, vs. RKIP-/- RKM 55±8%, p=0.02) while there was no effect in the SHAM group (WT SHAM 52±5%, RKIP-/- SHAM 48±6%). In addition, the number of circulating CD45⁺/collagen I⁺ fibrocytes (FACS) in the peripheral blood was increased by RKM and reduced by RKIP-knockout. Mechanistically, systemic RKIP-deficiency in CKD led to a two-fold increase of the nuclear myocardial accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2), the main transcriptional activator of antioxidative proteins. RKIP-/- RKM mice were also characterized by higher myocardial protein expression of the active form of endothelial nitric oxide synthase (eNOS) (phospho-S1177), normalized to total eNOS.

Conclusions: In uremia-induced myocardial maladaptive remodeling, systemic RKIP-deficiency improves left ventricular fibrosis and cardiomyocyte hypertrophy, associated with a reduction of myocardial oxidative stress, probably through the increased myocardial accumulation of nuclear Nrf2 and enhanced myocardial protein expression of the active form of eNOS (phospho-S1177).