“Avaliação do efeito da reposição de αklotho recombinante sobre a remodelação miocárdica em modelo experimental de doença renal crônica”

Abstract

ntroduction: Chronic Kidney Disease (CKD) related cardiomyopathy - Uremic Cardiomyopathy - is the underlying reason that makes Sudden Cardiac Death the main mortality cause on dialysis. Among the mechanisms involved in its pathophysiology, the Bone and Mineral Disorder of CKD (BMD-CKD) stands out doe to the presence of hyperphosphatemia, elevated levels of FGF23 and low levels of αKlotho. Evidence suggests that αKlotho replacement would have a protective effect on the myocardium, but the mechanisms are still poorly understood. Objective: To evaluate, in an experimental model of CKD, the effect of recombinant αKlotho replacement on cardiac remodeling and potential pathophysiological mechanisms involved. Methods: We studied 46 male Wistar rats with 12 weeks, divided into 3 groups: Control (sham), DRC and DRC + KL. The animals were submitted to surgery (sham or nephrectomy 5/6) and treated from the 4th to 8th week with recombinant αKlotho (0.01mg / kg, SC, 48 / 48h) or vehicle. At week 8, echocardiography and creatinine, urea, calcium, phosphorus, PTHi, FGF23, FGF21 and sKlotho were measured. Myocardial remodeling was evaluated by cardiac mass, echocardiographic parameters and myocardial histomorphometry. Myocardial expression of MHCβ and αSMA were assessed by immunohistochemistry (IHC) and qRT-PCR, while the effect on myocardial expression of TRPC6 and FGF21 was determined by IHC, westernblot and qRT-PCR. Results: There was an increase in creatinine (Control: 0.24 ± 0.04 vs CKD: 0.58 ± 0.07 and CKD+KL: 0.62 ± 0.06mg / dl, p <0.0001) and drop in its clearance (Control: 2.0 ± 0.5 vs CKD: 1.1 ± 0.13 and CKD+KL: 1.0 ± 0.13 ml / min / 100g; p <0.0001) in the groups with CKD. In the CKD groups, there was an increase in the excreted fraction of phosphorus (Control: 8.9 ± 1.6 vs CKD: 20.08 ± 3.8 and CKD+KL: 24.2 ± 5.3%, p < 0.0001); (P <0.05) and FGF23 (Control: 129.9 ± 28.7 vs. CKD: 208.8 ± 33.9 and CKD+KL: 200.5 ± 32.0 mg / dL, p <0.05), and reduction of serum αKlotho levels (Control: 188.4 ± 29.67 vs. CKD: 36.2 ± 21.3 and CKD+KL: 49.2 ± 25.6 pM, p <0.0001). In the assessment of myocardial remodeling, an increase in the heart weight/tibia length ratio (Control: 16.7 ± 1.0 vs. CKD: 20.2 ± 1.0 mg / mm, p <0.0001) and LV walls thickness: septum (Control: 1.41 ± 0.1 vs CKD: 1.58 ± 0.11 mm, p = 0.006), posterior wall (Control: 1.45 ± 0.1 vs CKD: 1.54 ± 0.1mm ; p = 0.01) and estimated LV mass (Control: 736.5 ± 123.4 vs CKD: 883.1 ± 128.4 mg; p = 0.005) in the CKD group, but with a reduction in the αKlotho treated group: heart/tibia (CKD: 20.2 ± 1.0 vs CKD+KL: 17.8 ± 0.9 mg/mm, p <0.0001), septum (CKD: 1.58 ± 0.11 vs. CKD+KL: 1.42 ± 0.10 mm, p = 0.006), posterior wall (CKD: 1.54 ± 0.1 vs CKD+KL: 1.42 ± 0.1mm, p = 0.01) and mass of VE (CKD: 883.1 ± 128.4 vs. CKD+KL: 750.4 ± 114.8mg, p = 0.005). It was also observed a reduction of cardiomyocyte diameter (CKD: 21.4 ± 0.8 vs. CKD+KL: 17.7 ± 1.0 μm, p <0.0001) and of interstitial fibrosis area (CKD: 4, 0 ± 0.53 vs. CKD+KL: 2.35 ± 0.3%, p <0.0001) in the treated group compared to untreated CKD group, as well as reduction in expression of MHCβ (IHC) (CKD: 28, 4 ± 2.4 vs. CKD+KL: 22.8 ± 4.0%, p = 0.002) and αSMA (IHC) (CKD: 7.2 ± 2.7 vs. CKD+KL: 2.6 ± 0.4 %, p = 0.0001). There was a reduction in the treated group of TRPC6 expression on cardiomyocyte membrane surface (CKD: 15.5 ± 3.72 vs. CKD+KL: 9.77 ± 1.84% area; p = 0.001) and increased FGF21 myocardial expression at mRNA (CKD: 0.73 ± 0.19 vs. CKD+KL: 0.96 ± 0.26; 2ΔΔCT, p = 0.03) and at protein level (CKD: 1.25 ± 0.11 vs. CKD+KL: 1.46 ± 0.22, UA, p = 0.03) in relation to the untreated CKD group. Conclusion: We conclude that, in an experimental model of CKD with moderate loss of renal function and cardiac hypertrophy, the replacement of rKlotho, at the dose and route used and therapeutic rationale, showed cardioprotective effect, attenuating myocardial remodeling. The mechanism of this effect involved not only the reduction of TRPC6 expression in the cardiomyocyte membrane, but also an increase in the myocardial expression of FGF21, which suggests the existence of an effect of sKlotho on its expression.