Uremic cardiac hypertrophy is reversed by rapamycin but not by lowering of blood pressure
2009; Elsevier BV; Volume: 75; Issue: 8 Linguagem: Inglês
10.1038/ki.2008.690
ISSN1523-1755
AutoresAndrew M. Siedlecki, Xiaohua Jin, Anthony J. Muslin,
Tópico(s)Ion Transport and Channel Regulation
ResumoChronic kidney disease is often complicated by uremic cardiomyopathy that consists of left ventricular hypertrophy and interstitial fibrosis. It is thought that hypertension and volume overload are major causes of this disease, but here we sought to identify additional mechanisms using a mouse model of chronic renal insufficiency. Mice with a remnant kidney developed an elevated blood urea nitrogen by 1 week, as expected, and showed progressive cardiac hypertrophy and fibrosis at 4 and 8 weeks even though their blood pressures were not elevated nor did they show signs of volume overload. Cardiac extracellular signal-regulated kinase (ERK) was activated in the uremic animals at 8 weeks. There was also an increased phosphorylation of S6 kinase, which is often mediated by activation of the mammalian target of rapamycin (mTOR). To test the involvement of this pathway, we treated these uremic mice with rapamycin and found that it reduced cardiac hypertrophy. Reduction of blood pressure, however, by hydralazine had no effect. These studies suggest that uremic cardiomyopathy is mediated by activation of a pathway that involves the mTOR pathway. Chronic kidney disease is often complicated by uremic cardiomyopathy that consists of left ventricular hypertrophy and interstitial fibrosis. It is thought that hypertension and volume overload are major causes of this disease, but here we sought to identify additional mechanisms using a mouse model of chronic renal insufficiency. Mice with a remnant kidney developed an elevated blood urea nitrogen by 1 week, as expected, and showed progressive cardiac hypertrophy and fibrosis at 4 and 8 weeks even though their blood pressures were not elevated nor did they show signs of volume overload. Cardiac extracellular signal-regulated kinase (ERK) was activated in the uremic animals at 8 weeks. There was also an increased phosphorylation of S6 kinase, which is often mediated by activation of the mammalian target of rapamycin (mTOR). To test the involvement of this pathway, we treated these uremic mice with rapamycin and found that it reduced cardiac hypertrophy. Reduction of blood pressure, however, by hydralazine had no effect. These studies suggest that uremic cardiomyopathy is mediated by activation of a pathway that involves the mTOR pathway. Pathological cardiac hypertrophy is associated with a poor prognosis, the development of cardiac arrhythmias, diastolic dysfunction, and progression to overt heart failure.1.London G.N. Cardiovascular disease in chronic renal failure: pathophysiologic aspects.Semin Dial. 2003; 16: 85-94Crossref PubMed Scopus (253) Google Scholar A similar condition develops in patients with severe chronic kidney disease (CKD) that is called uremic cardiomyopathy.1.London G.N. Cardiovascular disease in chronic renal failure: pathophysiologic aspects.Semin Dial. 2003; 16: 85-94Crossref PubMed Scopus (253) Google Scholar, 2.Foley R.N. Parfrey P.S. Harnett J.D. et al.The prognostic importance of left ventricular geometry in uremic cardiomyopathy.J Am Soc Nephrol. 1995; 5: 2024-2031PubMed Google Scholar, 3.Levin A. Singer J. Thompson C.R. et al.Prevalent left ventricular hypertrophy in the predialysis population: identifying opportunities for intervention.Am J Kidney Dis. 1996; 27: 347-354Abstract Full Text PDF PubMed Scopus (653) Google Scholar, 4.Stewart G.A. Gansevoort R.T. Mark P.B. et al.Electrocardiographic abnormalities and uremic cardiomyopathy.Kidney Int. 2005; 67: 217-226Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar In this condition, cardiomyocytes enlarge and excess fibrous tissue is deposited in the heart resulting in the thickening of the ventricular walls. Uremic cardiomyopathy is associated with diastolic dysfunction and also, in some cases, with contractile abnormalities. Nearly 75% of adults have left ventricular hypertrophy (LVH) at the time of initiation of dialysis for end-stage renal disease.5.Levin A. Thompson C.R. Ethier J. et al.Left ventricular mass index increase in early renal disease: impact of decline of hemoglobin.Am J Kidney Dis. 1999; 34: 125-134Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar The development of LVH is an independent risk factor that is associated with reduced survival in patients receiving dialysis.6.Silberberg J. Barre P.E. Prichard S.S. et al.Impact of left ventricular hypertrophy on survival in end-stage renal disease.Kidney Int. 1989; 36: 286-290Abstract Full Text PDF PubMed Scopus (748) Google Scholar The pathogenesis of uremic cardiomyopathy remains uncertain. As there is a very high incidence of hypertension in patients with severe CKD, one hypothesis is that cardiac hypertrophy develops as a result of pressure overload. However, correction of hypertension in rats with renal injury does not prevent the development of cardiac hypertrophy.7.Rambausek M. Ritz E. Mall G. et al.Myocardial hypertrophy in rats with renal insufficiency.Kidney Int. 1985; 28: 775-782Abstract Full Text PDF PubMed Scopus (88) Google Scholar Furthermore, Ayus et al.8.Ayus J.C. Mizani M. Achinger S.G. et al.Effects of short daily versus conventional hemodialysis on left ventricular hypertrophy and inflammatory markers: a prospective, controlled study.J Am Soc Nephrol. 2005; 16: 2778-2788Crossref PubMed Scopus (239) Google Scholar showed that ventricular wall dimensions were reduced in patients' receiving intensive daily dialysis for 1 year when compared with those receiving dialysis three times per week, despite similar systolic blood pressures in both groups. Volume overload may also contribute to LVH by increasing LV end-diastolic pressure.1.London G.N. Cardiovascular disease in chronic renal failure: pathophysiologic aspects.Semin Dial. 2003; 16: 85-94Crossref PubMed Scopus (253) Google Scholar, 9.Yamakawa H. Imamura T. Matsuo T. et al.Diastolic wall stress and ANG II in cardiac hypertrophy and gene expression induced by volume overload.Am J Physiol Heart Circ Physiol. 2000; 279: H2939-H2946PubMed Google Scholar A reduction in intradialytic weight correlates with a reduction in LV mass index, but LVH may persist after normalization of wall stress.9.Yamakawa H. Imamura T. Matsuo T. et al.Diastolic wall stress and ANG II in cardiac hypertrophy and gene expression induced by volume overload.Am J Physiol Heart Circ Physiol. 2000; 279: H2939-H2946PubMed Google Scholar Anemia is frequently present in patients with CKD and is another factor implicated in the pathogenesis of uremic cardiomyopathy.1.London G.N. Cardiovascular disease in chronic renal failure: pathophysiologic aspects.Semin Dial. 2003; 16: 85-94Crossref PubMed Scopus (253) Google Scholar, 10.Harnett J.D. Kent G.M. Foley R.N. et al.Cardiac function and hematocrit level.Am J Kidney Dis. 1995; 25: S3-S7Abstract Full Text PDF PubMed Scopus (138) Google Scholar An additional possibility is that the accumulation of hypertrophic ligands associated with renal insufficiency or end-stage renal disease may initiate a signal transduction cascade independent of mechanical stress. A variety of other substances accumulate in end-stage renal disease that may modulate cardiac growth and function, including endothelin-1, parathyroid hormone, tumor necrosis factor-α, leptin, interleukin-1α, and interleukin-6.11.Winchester J.F. Audia P.F. Extracorporeal strategies for the removal of middle molecules.Semin Dial. 2006; 19: 110-114Crossref PubMed Scopus (34) Google Scholar The molecular pathways responsible for cardiac hypertrophy are dependent on their initiating stimuli. In the setting of load-induced hypertrophy, mechanical stress can be translated through a detection mechanism that is hypothesized to be based on integrins that initiate intracellular signaling in response to stretch of the extracellular matrix.12.Yutao X. Geru W. Xiaojun B. et al.Mechanical stretch-induced hypertrophy of neonatal rat ventricular myocytes is mediated by beta(1)-integrin-microtubule signaling pathways.Eur J Heart Fail. 2006; 8: 16-22Crossref PubMed Scopus (29) Google Scholar Stretch may also promote the local release of ligands such as angiotensin II and endothelin-1 that bind to cognate receptors on the surface of cardiomyocytes to stimulate intracellular signaling pathways.13.Yamazaki T. Komuro I. Kudoh S. et al.Angiotensin II partly mediates mechanical stress-induced cardiac hypertrophy.Circ Res. 1995; 77: 258-265Crossref PubMed Scopus (265) Google Scholar, 14.Yamazaki T. Komuro I. Kudoh S. et al.Endothelin-1 is involved in mechanical stress-induced cardiomyocyte hypertrophy.J Biol Chem. 1996; 271: 3221-3228Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar Subsequent activation of the mammalian target of rapamycin (mTOR) complex activates the ribosomal S6 kinase that promotes mRNA translation.15.Proud C.G. Ras, PI3-kinase and mTOR signaling in cardiac hypertrophy.Cardiovasc Res. 2004; 63: 403-413Crossref PubMed Scopus (140) Google Scholar It is hypothesized that uremic cardiomyopathy may occur through a separate mechanism that is initiated by activation of Na/K ATPase channels. Cardiotonic steroids accumulate in patients with CKD and they interact with the α subunit of transmembrane Na/K ATPases to promote the activation of the intracellular mitogen-activated protein kinase pathway.16.Kennedy D.J. Vetteth S. Periyasamy S.M. et al.Central role for the cardiotonic steroid marinobufagenin in the pathogenesis of experimental uremic cardiomyopathy.Hypertension. 2006; 47: 488-495Crossref PubMed Scopus (217) Google Scholar, 17.Schoner W. Scheiner-Bobis G. Endogenous and exogenous cardiac glycosides: their roles in hypertension, salt metabolism, and cell growth.Am J Physiol Cell Physiol. 2007; 293: C509-C536Crossref PubMed Scopus (387) Google Scholar Rapamycin, a direct inhibitor of mTOR reverses hypertrophy due to chronic pressure overload but has not been investigated in CKD.18.Shioi T. McMullen J.R. Tarnavski O. et al.Rapamycin attenuates load-induced cardiac hypertrophy in mice.Circulation. 2003; 107: 1664-1670Crossref PubMed Scopus (393) Google Scholar mTOR acts through several downstream effectors, including S6 Kinase, 4E-BP, and EF1α, that all modulate ribosomal function. The PI3 kinase-Akt1 pathway and the extracellular signal-regulated kinase (ERK) pathway both act upstream of mTOR to promote increased protein synthesis.19.Ma L. Teruya-Feldstein J. Bonner P. et al.Identification of S664 TSC2 phosphorylation as a marker for extracellular signal-regulated kinase mediated mTOR activation in tuberous sclerosis and human cancer.Cancer Res. 2007; 67: 7106-7112Crossref PubMed Scopus (122) Google Scholar In this work, we established a murine model of pressure-controlled uremic cardiomyopathy and identified mTOR as a key protein in the development of this condition. To determine whether CKD in mice results in the development of cardiac hypertrophy, we studied the effect of surgically induced renal injury (SIRI) on 12-week-old 129/SvJ mice. In this method, the right kidney was injured by cauterization, and a left nephrectomy was performed after 2 weeks. The surgery was well tolerated by wild type 129/SvJ mice and no mortality was observed. The metabolic status of mice was determined both 4 and 8 weeks after the completion of SIRI or sham surgery (Table 1). The serum creatinine was significantly increased in SIRI mice compared with sham animals at both 4- and 8-week time points. Four weeks after surgery, the serum creatinine was 0.71±0.006 in sham animals but was increased to 0.133±0.015 in SIRI mice (P=0.003; Table 1). Eight weeks after surgery, the serum creatinine was 0.104±0.007 in sham mice but was increased 30.7% to 0.136±0.018 in SIRI mice (P=0.016). Similarly, the BUN was significantly increased in SIRI mice at both the 4- and 8-week time points (Table 1). Four weeks after surgery, the serum BUN was 16.0±4.1 mg/100 ml in sham mice but was increased to 54.0±4 mg/dl in SIRI animals (P=0.00003). Eight weeks after surgery, the serum BUN was 22±1 mg/100 ml in sham mice but was increased to 83±3 mg/dl in SIRI animals (P=0.0001). A three- to four-fold increase in urine output was noted in SIRI animals compared with sham-operated animals at both 4 and 8 weeks after surgery (Table 1). Serum aldosterone levels were significantly increased in SIRI mice compared with sham mice at both the 4- and 8-week time points (P=0.042), and this was associated with significantly elevated urine sodium loss (P=0.041). Serum angiotensin II levels were transiently elevated in SIRI mice compared with sham mice 4 weeks after surgery (P=0.013) but were significantly reduced in SIRI mice compared with sham mice at the 8-week time point (P=0.0001; Table 1).20.Chen L.Y. Li P. He Q. et al.Transgenic study of the function of chymase in heart remodeling.J Hypertens. 2002; 20: 2047-2055Crossref PubMed Scopus (28) Google Scholar, 21.Xu J. Carretero O.A. Lin C.X. et al.Role of cardiac overexpression of ANG II in the regulation of cardiac function and remodeling postmyocardial infarction.Am J Physiol Heart Circ Physiol. 2007; 293: H1900-H1907Crossref PubMed Scopus (39) Google Scholar Serum potassium was significantly decreased in SIRI mice to 3.9±0.1 mmol/l, compared with 4.7±0.2 mmol/l in sham-operated mice (P=0.01; Table 1).Table 1Metabolic analysis of mice after renal injurySham (n=7) 4 weeksSIRI (n=8) 4 weeksSham (n=7) 8 weeksSIRI (n=8) 8 weeksSIRI+V (n=7) 8 weeksSIRI+Rap (n=7) 8 weeksSer. creatinine (mg/100 ml)0.071±0.0060.133±0.015*P<0.05 vs sham-operated mice at the same time point.0.104±0.0070.136±0.018*P<0.05 vs sham-operated mice at the same time point.0.141±0.008*P<0.05 vs sham-operated mice at the same time point.0.175±0.022*P<0.05 vs sham-operated mice at the same time point.Ser. hemoglobin (g/100 ml)17±0.316±0.115±1.615±1.116±0.712±2.0*P<0.05 vs sham-operated mice at the same time point.BUN (mg/dl)16±454±4*P<0.05 vs sham-operated mice at the same time point.22±183±3*P<0.05 vs sham-operated mice at the same time point.84±6*P<0.05 vs sham-operated mice at the same time point.108±11*P<0.05 vs sham-operated mice at the same time point.24 h urine volume (ml)0.530±901.730±190*P<0.05 vs sham-operated mice at the same time point.1.110±603.420±180*P<0.05 vs sham-operated mice at the same time point.3.640±840*P<0.05 vs sham-operated mice at the same time point.6.422±930*P<0.05 vs sham-operated mice at the same time point.Urine [Na+] (mmol/l)151±1399±6.3*P<0.05 vs sham-operated mice at the same time point.162±19108±8.558±4.2*P<0.05 vs sham-operated mice at the same time point.48±1.5*P<0.05 vs sham-operated mice at the same time point.24 h urine Na+ (mmol)0.0800±0.020.173±0.05*P<0.05 vs sham-operated mice at the same time point.0.180±0.010.369±0.02*P<0.05 vs sham-operated mice at the same time point.0.211±0.02*P<0.05 vs sham-operated mice at the same time point.0.306±0.01*P<0.05 vs sham-operated mice at the same time point.Ser. potassium (mmol/l)4.7±0.23.9±0.1*P<0.05 vs sham-operated mice at the same time point.3.8±0.9*P<0.05 vs sham-operated mice at the same time point.4.2±1.0Ser. aldosterone (pmol/l)2454±1963328±266*P<0.05 vs sham-operated mice at the same time point.2745±2203408±273*P<0.05 vs sham-operated mice at the same time point.3631±290*P<0.05 vs sham-operated mice at the same time point.3663±293*P<0.05 vs sham-operated mice at the same time point.Ser. angiotensin II (pg/ml)34.3±4142±17*P<0.05 vs sham-operated mice at the same time point.128±1597.3±12*P<0.05 vs sham-operated mice at the same time point.189±2342.3±5*P<0.05 vs sham-operated mice at the same time point.Metabolic analysis of 20-week-old mice after sham surgery (Sham), surgically induced renal injury (SIRI), SIRI with daily injections of vehicle (SIRI+V), or SIRI with daily injections of rapamcyin (SIRI+Rap). Daily injections of vehicle or rapamycin were initiated 4 weeks after SIRI and were given for 4 weeks.* P<0.05 vs sham-operated mice at the same time point. Open table in a new tab Metabolic analysis of 20-week-old mice after sham surgery (Sham), surgically induced renal injury (SIRI), SIRI with daily injections of vehicle (SIRI+V), or SIRI with daily injections of rapamcyin (SIRI+Rap). Daily injections of vehicle or rapamycin were initiated 4 weeks after SIRI and were given for 4 weeks. The body weight did not change after surgery and was 25.1±0.6 g in SIRI mice compared with 24.7±0.9 g in sham animals at the 8-week time point (P=0.51; Table 2). Interestingly, SIRI mice ate 42.5% more food than sham-operated animals (Table 2). Despite their increased food intake, SIRI mice had similar body composition to sham-operated mice. Measurement of muscle and fat mass by MRI demonstrated that the lean body mass of SIRI mice was 19.2±1.2 g 8 weeks after surgery and was 20.3±0.8 g in sham-operated mice (P=0.47; Table 2). Finally, the extracellular fluid volume was nearly identical in SIRI and sham-operated mice 8 weeks after surgery (Table 2).Table 2Body mass components of mice after renal injurySham (n=7) 8 weeksSIRI (n=8) 8 weeksSIRI+V (n=7) 8 weeksSIRI+Rap (n=7) 8 weeksBody weight (g)24.7±0.8525.1±0.626.1±0.525.7±0.624 h calorie intake (kcal)14.1±0.7520.1±1.5*P<0.05 vs sham-operated mice at the same time point.14.1±1.020.4±1.3*P<0.05 vs sham-operated mice at the same time point.24 h water intake (ml)1.130±1863.599±757*P<0.05 vs sham-operated mice at the same time point.3.630±510*P<0.05 vs sham-operated mice at the same time point.6.333±646*P<0.05 vs sham-operated mice at the same time point.24 h urine volume (ml)1.110±603.420±180*P<0.05 vs sham-operated mice at the same time point.3.640±840*P<0.05 vs sham-operated mice at the same time point.6.422±930*P<0.05 vs sham-operated mice at the same time point.Lean body mass (g)20.3±0.819.2±1.220.3±1.419.9±1.3Fat body mass (g)4.1±0.33.9±0.24.4±0.24.3±0.2Extracellular fluid volume (ml)4.13±1.244.10±0.894.21±0.994.02±1.16Lung weight/BW (mg/g)6.45±0.96.21±0.96.27±0.0.56.59±0.6Liver weight/BW (mg/g)26.12±2.426.82±2.027.06±4.426.55±1.9Spleen weight/BW (mg/g)2.52±0.32.71±0.42.93±0.32.62±0.4Metabolic analysis of 20-week-old mice after sham surgery (Sham), surgically induced renal injury (SIRI), SIRI with daily injections of vehicle (SIRI+V), or SIRI with daily injections of rapamcyin (SIRI+Rap).* P<0.05 vs sham-operated mice at the same time point. Open table in a new tab Metabolic analysis of 20-week-old mice after sham surgery (Sham), surgically induced renal injury (SIRI), SIRI with daily injections of vehicle (SIRI+V), or SIRI with daily injections of rapamcyin (SIRI+Rap). To evaluate the development of cardiac hypertrophy after SIRI, sequential transthoracic echocardiography was performed on unanesthetized mice 4 and 8 weeks after SIRI or sham surgery. LV systolic function, measured as fractional shortening, was nearly identical in SIRI and sham-operated mice at both time points (Table 3). The echocardiographically determined LV mass index increased from 3.22±0.08 mg/g in sham-operated animals to 3.78±0.14 mg/g in SIRI animals 4 weeks after surgery was completed (P=0.003). Similarly, at 8 weeks after surgery, the LV mass index increased from 3.20±0.15 mg/g in sham-operated animals to 3.96±0.11 mg/g in SIRI animals (P=0.001).Table 3Echocardiographic analysis of mice after renal injurySham (n=12) 4 weeksSIRI (n=15) 4 weeksSham (n=7) 8 weeksSIRI (n=8) 8 weeksSIRI+V (n=7) 8 weeksSIRI+Rap (n=7) 8 weeksHR (bpm)613±58618±31629±12637±9599±12616±15PWd (mm)0.76±0.060.89±0.09*P<0.05 for all comparisons vs sham-operated mice at the same time point except LVMI, where P<0.0210.76±0.020.92±0.02*P<0.05 for all comparisons vs sham-operated mice at the same time point except LVMI, where P<0.0210.85±0.05*P<0.05 for all comparisons vs sham-operated mice at the same time point except LVMI, where P<0.0210.78±0.02†P<0.05 for all comparisons vs SIRI mice injected with vehicle at the same time point except LVMI, where P<0.021.IVSd (mm)0.83±0.020.94±0.03*P<0.05 for all comparisons vs sham-operated mice at the same time point except LVMI, where P<0.0210.84±0.021.03±0.03*P<0.05 for all comparisons vs sham-operated mice at the same time point except LVMI, where P<0.0210.90±0.030.89±0.04†P<0.05 for all comparisons vs SIRI mice injected with vehicle at the same time point except LVMI, where P<0.021.LVEDD (mm)3.3±0.083.2±0.063.4±0.093.2±0.063.2±0.073.0±0.04LVM (mg)84±2.899±4.2*P<0.05 for all comparisons vs sham-operated mice at the same time point except LVMI, where P<0.02188±3.7106±2.2*P<0.05 for all comparisons vs sham-operated mice at the same time point except LVMI, where P<0.02194±4.976.1±3.9†P<0.05 for all comparisons vs SIRI mice injected with vehicle at the same time point except LVMI, where P<0.021.LVMI (mg/g)3.22±0.083.78±.14*P<0.05 for all comparisons vs sham-operated mice at the same time point except LVMI, where P<0.0213.20±0.153.96±0.11*P<0.05 for all comparisons vs sham-operated mice at the same time point except LVMI, where P<0.0213.63±0.17*P<0.05 for all comparisons vs sham-operated mice at the same time point except LVMI, where P<0.0212.94±0.18†P<0.05 for all comparisons vs SIRI mice injected with vehicle at the same time point except LVMI, where P<0.021.FS (%)63±1.062±1.264±1.065±0.965±1.465±1.9HR, heart rate; PWd, left ventricular posterior wall thickness in diastole; IVSd, interventricular septal wall thickness in diastole; LVEDD, left ventricular end-diastolic diameter; LVM, calculated LV mass (mg); LVMI, left ventricular mass index (LVM/body weight) mg/g; FS, fractional shortening of the left ventricle in systole. Transthoracic echcardiographic analysis of mice 4 or 8 weeks after sham surgery (Sham), subtotal nephrectomy (SIRI), SIRI with daily injections of vehicle (SIRI+V), or SIRI with daily injections of rapamcyin (SIRI+Rap). Daily injections of vehicle or rapamycin were initiated 4 weeks after SIRI and were given for 4 weeks.* P<0.05 for all comparisons vs sham-operated mice at the same time point except LVMI, where P<0.021† P<0.05 for all comparisons vs SIRI mice injected with vehicle at the same time point except LVMI, where P<0.021. Open table in a new tab HR, heart rate; PWd, left ventricular posterior wall thickness in diastole; IVSd, interventricular septal wall thickness in diastole; LVEDD, left ventricular end-diastolic diameter; LVM, calculated LV mass (mg); LVMI, left ventricular mass index (LVM/body weight) mg/g; FS, fractional shortening of the left ventricle in systole. Transthoracic echcardiographic analysis of mice 4 or 8 weeks after sham surgery (Sham), subtotal nephrectomy (SIRI), SIRI with daily injections of vehicle (SIRI+V), or SIRI with daily injections of rapamcyin (SIRI+Rap). Daily injections of vehicle or rapamycin were initiated 4 weeks after SIRI and were given for 4 weeks. To confirm whether SIRI promoted cardiac hypertrophy, we weighed heart tissue of SIRI and control mice 8 weeks after surgery (Table 4). The biventricular weight-to-tibial length ratio was increased from 6.3±0.1 mg/mm in sham-operated mice to 7.4±0.3 mg/mm in SIRI animals (P=0.012). Furthermore, the LV weight-to-tibial length ratio increased from 5.0±0.18 mg/mm sham mice compared with 5.9±0.22 mg/mm in SIRI mice (P=0.006; Table 4).Table 4Heart weights of mice after renal injurySham (n=7)SIRI (n=8)SIRI+V (n=7)SIRI+Rap (n=7)BW (g)24.7±1.425.4±1.126.1±0.925.7±1.3BVW (mg)123±2.7149±6.5*P<0.025 vs sham-operated mice138±4.1114±3.9†P<0.021 vs SIRI mice injected with vehicle.LV (mg)96.5±3.2119±4.9*P<0.025 vs sham-operated mice113±3.490.2±3.9†P<0.021 vs SIRI mice injected with vehicle.RV (mg)25.8±1.731.0±1.0*P<0.025 vs sham-operated mice29.8±2.823.7±0.9†P<0.021 vs SIRI mice injected with vehicle.Tibia (TL) (cm)1.94±0.021.99±0.022.02±0.021.98±0.02BVW/BW (mg/g)4.98±0.25.9±0.2*P<0.025 vs sham-operated mice5.3±0.14.4±0.1†P<0.021 vs SIRI mice injected with vehicle.BVW/TL (mg/mm)6.3±0.17.4±0.3*P<0.025 vs sham-operated mice6.8±0.25.8±0.2†P<0.021 vs SIRI mice injected with vehicle.LV/BW (mg/g)3.9±0.14.7±0.2*P<0.025 vs sham-operated mice4.3±0.13.8±0.3†P<0.021 vs SIRI mice injected with vehicle.LV/TL (mg/mm)5.0±0.25.9±0.2*P<0.025 vs sham-operated mice5.6±0.24.6±0.2†P<0.021 vs SIRI mice injected with vehicle.RV/BW (mg/g)1.2±0.01.2±0.00.9±0.11.0±0.1RV/TL (mg/mm)1.5±0.11.5±0.21.3±0.11.2±0.1BW, body weight; BVW, biventricular weight; LV, left ventricular weight; RV, right ventricular weight; Tibia, tibial length; BVW/BW, biventricular weight-to-body weight ratio; BVW/TL, biventricular weight-to-tibial length ratio; LV/BW, left ventricular weight-to-body weight ratio; LV/TL, left ventricular weight-to-tibial length ratio; RV/BW, right ventricular weight-to-body weight ratio; RV/TL, right ventricular weight-to-tibial length ratio. Morphometric analysis of 20-week-old mice 8 weeks after sham surgery (Sham), subtotal nephrectomy (SIRI), SIRI with daily injections of vehicle (SIRI+V), or SIRI with daily injections of rapamcyin (SIRI+Rap). Daily injections of vehicle or rapamycin were initiated 4 weeks after SIRI and were given for 4 weeks.* P<0.025 vs sham-operated mice† P<0.021 vs SIRI mice injected with vehicle. Open table in a new tab BW, body weight; BVW, biventricular weight; LV, left ventricular weight; RV, right ventricular weight; Tibia, tibial length; BVW/BW, biventricular weight-to-body weight ratio; BVW/TL, biventricular weight-to-tibial length ratio; LV/BW, left ventricular weight-to-body weight ratio; LV/TL, left ventricular weight-to-tibial length ratio; RV/BW, right ventricular weight-to-body weight ratio; RV/TL, right ventricular weight-to-tibial length ratio. Morphometric analysis of 20-week-old mice 8 weeks after sham surgery (Sham), subtotal nephrectomy (SIRI), SIRI with daily injections of vehicle (SIRI+V), or SIRI with daily injections of rapamcyin (SIRI+Rap). Daily injections of vehicle or rapamycin were initiated 4 weeks after SIRI and were given for 4 weeks. To evaluate whether SIRI promoted the growth of individual cardiomyocytes and the deposition of extracellular matrix in cardiac tissue, we completed a histologic analysis of ventricular tissue sections. Cardiomyocyte area increased by 119% in SIRI mice when compared with sham-operated animals 8 weeks after surgery (P=0.0001; Figure 1). Furthermore, cardiac fibrosis, evaluated in Masson's trichrome-stained ventricular sections, increased by 120% in SIRI mice 8 weeks after surgery (P=0.0001; Figure 2).Figure 2Evaluation of cardiac fibrosis in ventricular tissue obtained 8 weeks after surgery. Ventricular tissue was fixed, embedded, sectioned at 4-micron intervals, stained with Masson's trichrome, and examined by light microscopy. (a) Ventricular section from sham-operated mouse. (b) Ventricular tissue section from surgically induced renal injury (SIRI) mouse. (c) Section from SIRI mouse treated with vehicle (V) for 4 weeks. (d) Section from SIRI mouse treated with rapamycin (R) for 4 weeks. (e) Quantitative evaluation of fibrotic area/total area by computerized analysis of ventricular tissue sections. Three selected high power fields (400 ×) from each of the 4 animals per group were analyzed by computerized photomicrography with ImageJ v1.24 software. Fibrotic area was calculated by use of the ImageJ Particle Analyzer algorithm. *P<0.05 vs sham.View Large Image Figure ViewerDownload (PPT) To better evaluate cardiac systolic and diastolic function, we performed cardiac catheterization on anesthetized SIRI and control mice 8 weeks after surgery was completed. These studies demonstrated that LV systolic function, measured by determining +dP/dTmax, was unchanged in SIRI mice (8844±667 mm Hg/s) when compared with sham-operated mice (8758±856 mm Hg/s) 8 weeks after surgery (Table 5). Furthermore, diastolic function, measured as τ (Glantz), was similar in both SIRI and sham-operated mice 8 weeks after surgery. Systolic blood pressure, measured by determining the peak LV systolic pressure, was 139±11 mm Hg in sham-operated mice and was similar at 141±12 mm Hg in SIRI mice (P=NS). Furthermore, SIRI mice were found to be normotensive by tail cuff blood pressure measurements and also by determination of carotid artery pressures with a Millar catheter (Table 5).Table 5Invasive hemodynamic analysis of mice after renal injury and rapamycin treatmentSham (n=7)SIRI (n=8)SIRI+V (n=7)SIRI+R (n=7)HR (bpm)457±12453±10439±12439±11LVPmax (mm Hg)139±11141±12132±12125±9LVEDP (mm Hg)5.3±1.55.5±0.84.3±0.713.6±0.9Tau (Glantz)11.8±0.412.5±1.512.0±0.814.2±1.4+dP/dtmax (mm Hg/s)8758±8568752±6938971±1007624±956Systolic TCP (mm Hg)129±4123±5124±4127±2Diastolic TCP (mm Hg)82±375±471±373±4Systolic CAP (mm Hg)133±11134±10138±15144±14HR, heart rate obtained during cardiac catheterization; LVPmax, peak systolic LV pressure; LVEDP, left ventricular end-diastolic pressure; Tau, time constant of isovolumic change; +dP/dtmax, the maximal change in LV pressure per unit time; TCP, tail cuff pressure; CAP, carotid artery pressure. Invasive hemodynamic analysis of 20-week-old mice 8 weeks after sham surgery (Sham), subtotal nephrectomy (SIRI), subtotal nephrectomy+vehicle (SIRI+V), or subtotal nephrectomy+rapamycin (SIR+R) by cardiac catheterization. Open table in a new tab HR, heart rate obtained during cardiac catheterization; LVPmax, peak systolic LV pressure; LVEDP, left ventricular end-diastolic pressure; Tau, time constant of isovolumic change; +dP/dtmax, the maximal change in LV pressure per unit time; TCP, tail cuff pressure; CAP, carotid artery pressure. Invasive hemodynamic analysis of 20-week-old mice 8 weeks after sham surgery (Sham), subtotal nephrectomy (SIRI), subtotal nephrectomy+vehicle (SIRI+V), or subtotal nephrectomy+rapamycin (SIR
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