Background: Chronic kidney disease (CKD) leads to cardiac remodeling and eventually heart failure. We studied the role of myocardial endothelial nitric oxide synthase (eNOS), the renin-angiotensin-system (RAS) and blood pressure in cardiac fibrogenesis in CKD mice.

Methods & Results: 10-week-old male C57/Bl6N (BL6) and SV129 (a strain with 2 renin genes) mice underwent 5/6 nephrectomy (remnant kidney model, RKM) or sham operation for 10 weeks. RKM significantly enhanced plasma creatinine and urea. Histological analyses (picrosirius red staining) demonstrated both renal and left ventricular (LV) fibrosis, pronounced in SV129 mice (BL6: SHAM 5.6±0.4%, RKM 7.3±0.7%, p=0.04; SV129: SHAM 6.0±0.5%, RKM 14±2%, p=0.001). Likewise, RKM increased the number of LV fibroblasts/mm² (BL6: SHAM 36±4, RKM 85±13, p=0.001; SV129: SHAM 82±11, RKM 200±34, p=0.006) and cardiac protein expression of fibrosis regulators fibronectin and CTGF.

Mechanistically, CKD down-regulated cardiac expression of the active form of eNOS (phospho-S1177) and differently regulated expression of RAS components. In SV129 mice, prorenin (RKM 142±14 %SHAM, p=0.02) and AngII (SV129: RKM 159±28 %SHAM, p=0.04) expression were significantly increased. Renin 1 was up-regulated in BL6 (BL6: RKM 142±16 %SHAM, p=0.04). Oxidative stress as measured by co-immunostaining for intracellular fibronectin and 8-hydroxyguanosine and the percentage of CXCR4⁺ fibroblasts in the LV myocardium were increased by RKM. In addition, the number of circulating CD45⁺ / collagen I⁺ fibrocytes (FACS) in the peripheral blood was increased by RKM in BL6 (SHAM 671±178, RKM 1203±225/10⁴ cells, p=0.04) and reduced in SV129 animals (SHAM 1182±275, RKM 507±149/10⁴ cells, p=0.04). In BL6 mice transplanted with green fluorescent protein GFP⁺ bone marrow, RKM increased the percentage of GFP⁺ fibroblasts in kidney and heart to 13-20%. CKD significantly reduced LV capillarization and increased apoptosis of cardiomyocytes.

RKM was associated with a modest increase of peripheral blood pressure (Ø 30mmHg) but normalization with hydralazine (250 mg/L/day) did not diminish cardiac fibrogenesis in both strains.

Adult cardiac fibroblasts isolated from BL6 mice were treated with 10% healthy vs. uremic serum of patients with terminal CKD for 24 h. Uremic serum uncoupled eNOS (monomers/dimers: healthy 0.8±0.2 IOD vs. uremic serum 2.3±0.3 IOD, p=0.03) and significantly increased protein expression of fibronectin, CTGF and pro-inflammatory inducible NOS.

Conclusions:  Chronic kidney disease in the murine remnant kidney model is associated with cardiac fibrosis, especially in the context of a second hit (renin-overexpressing SV129 mice). The mechanisms of fibrogenesis are independent of blood pressure and involve mobilization of fibroblasts from the bone marrow, the SDF1/CXCR4 axis, oxidative stress, and cardiomyocyte apoptosis, which are governed by RAS activation and eNOS uncoupling.