Effects Of Klotho On Attenuating The Left Ventricular Hypertrophy Induced By Uremic Toxins

Background:Uremic cardiomyopathy is the most common complication of chronic kidney disease (CKD), and results in a higher mortality in these patients. Left ventricular hypertrophy (LVH), the prominent pathological feature of uremic cardiomyopathy, is usually caused by hypertension and increased volume load. However, in patients with CKD, it is not possible to control the progression of LVH via controlling blood pressure and lowering volume load. Recently, extensive studies have been performed on the relationship between internal environmental changes and cardiovascular complications in CKD patients. In particular, oxidative stress, a comparatively prominent factor in the internal environment, has been approved to play a crucial role in inducing LVH. Unlike the normal individuals, patients with CKD are in a state of long-term, high oxidative stress, which is associated with accumulation of toxins following renal function decline, particularly protein binding toxins such as indoxyl sulfate (IS). These toxins can stimulate overproduction of reactive oxygen species (ROS) and contribute to the elevation of oxidative stress. As the toxins cannot be removed by conventional dialysis, the toxins accumulate in blood circulation, which finally resulting in severe impairment of cardiovascular system. On this basis, we speculate that the uremic toxins may contribute to the development and progression of uremic myocardiopathy through up-regulating oxidative stress. Therefore, understanding the roles of uremic toxins in inducing LVH may be helpful in discovering the pathogenesis of uremic myocardiopathy, based on which to select efficient strategies for the prevention and treatment of LVH.Klotho, an anti-aging gene, is specifically expressed in few organs such as kidney, choroid and reproductive organs. Secreted form of Klotho protein (secKlotho) can penetrate into blood circulation, and function as a humoral factor. It has been demonstrated that the anti-aging property of Klotho is related with its strong anti-oxidative stress effect. For the mice with over-expression of Klotho gene, the life span is prolonged, and the production of ROS is decreased significantly. In addition, studies have shown that secKlotho can promote the production of superoxide dismutase (SOD), based on which to accelerate the removal of ROS. Further, in hypertensive rats transfected with Klotho, the expression of NADPH oxidase2(NOX2) in kidney and aorta is inhibited and the generation of superoxide is decreased, delaying the impairments to renal and vascular functions. Nowadays, although the mechanism of oxidative stress modulated by Klotho is still not completely understood, secKlotho has been approved as an important endogenous protein responsible for anti-oxidant stress considering its role in inhibiting the generation of ROS and scavenging of excessive ROS.Mice with Klotho defect are apt to develop cardiovascular diseases and death within a short time, which is similar to patients with uremia. Thus, an increasing number of studies have focus on the relationship between Klotho and cadiovascular complications in chronic kidney diseases. Our study demonstrated that, in mice with uremia, the expression of Klotho was significantly attenuated in kidney and serum. Nevertheless, the atherosclerotic lesions were improved obviously in mice with uremia transfected with Klotho. This indicated Klotho protein was decreased significantly in patients with CKD. On this basis, we speculate that up-regulation of Klotho may improve the vascular lesions in patients with CKD. In the past few years, studies found that significant LVH was developed in Klotho knockout mice, which indicated that Klotho was associated with the development and progression of LVH. Therefore, we supposed that uremic toxins could induce LVH through improving oxidative stress in CKD. Meanwhile, renal lesions could lead to decreased expression and secretion of Klotho protein, based on which the capacity of endogenous anti-oxidant stress is decreased and inhibition of uremic toxin-induced LVH was attenuated. The imbalance of oxidation and anti-oxidation was likely to induce high incidence and rapid progression of LVH. Further, up-regulation of Klotho or ectogenous Klotho can inhibit the development and progression of LVH through increasing anti-oxidant stress levels, based on which to improve the prognosis of uremic myocardiopathy.In this study, we aim to investigate the effects of IS and recombinant human Klotho protein on the induction of LVH in myocardial cells derived from neonate rat in vitro, as well as uremic mice model and Klotho gene knockout heterozygote (kl/+) mice in vivo. To discover the potential role of Klotho in IS-induced LVH, we focus on:①the roles of IS in inducing myocardial hypertrophy;②the effects of Klotho on myocardial hypertrophy induced by IS;③the effects of IS on LVH in kl/+mice and normal group;④establishing a uremic mice model, through which to identify the effects of Klotho (via intraperitoneal injection) on LVH, and confirm whether ectogenous Klotho could relieve uremic LVH.Results:IS injection decreases the expression of renal Klotho and induces more severe LVH in kl/+miceTo further evaluate the relationships between IS, Klotho and LVH, we treated C57BL/6J mice directly with IS (100mg/kg/d) by intraperitoneal injection for8weeks and found that IS treatment resulted in an approximate5.0-fold increase of IS in serum. The increase in serum IS was accompanied by a significant down-regulation of Klotho expression in the kidneys. Notably, an obvious increase in the ventricular wall thickness, elevation of relative heart weight, as well as the decrease in left ventricular internal diameter diastole and increase in left ventricular posterior wall thickness diastole were observed in IS-injected WT and kl/+mice. Interestingly, relative to wide type mice, kl/+mice developed more severe LVH after injection with IS for8weeks. These results indicate that a high level of circulating IS is a causal factor for LVH, and the decrease of Klotho is probably implicated in IS induced LVH.Klotho has the ability to inhibit IS-induced cardiomyocyte hypertrophyTo reveal the direct effect of IS on cardiomyocyte hypertrophy and whether Klotho has a counteractive action against IS, we then treated the cultured NRCMs with IS in the absence or presence of Klotho. Firstly, it was found that IS could dose-dependently promote neonate rat cardiac myocytes (NRCMs) uptake of3H-leucine. Moreover, the cells became obviously enlarged after being cultured with500μmol/L IS for48h, accompanied by significant increases in the mRNA levels of ANF, BNP and β-MHC, demonstrating that IS has an inductive effect on cardiomyocyte hypertrophy. Notably, pre-treatment with400pmol/L Klotho protein for1h significantly inhibited IS-induced cardiomyocyte hypertrophy, suggesting that Klotho has a distinct ability to suppress the toxic action of IS on cardiomyocytes. Repression of oxidative stress and its downstream signaling pathways contributes to Klotho inhibition of IS-induced cardiomyocyte hypertrophySince IS is characterized with oxidative property and oxidative stress plays an important role in myocardial hypertrophy, we next investigated the role of oxidative stress in IS-induced cardiomyocyte hypertrophy. As expected, IS could quickly promote ROS production in NRCMs and incubation with500μmol/L of IS for10min led to an about2-fold increase in ROS level. Notably, pretreatment with Klotho could dose-dependently suppress IS-induced ROS production. It was further found that the expressions of NOX2and NOX4were significantly up-regulated in cardiomyocytes after500μmol/L IS treatment, while pretreatment with400pmol/L Klotho protein could inhibit IS-induced up-regulation of NOX2and NOX4, similar to the effect of DPI (an NADPH oxidase inhibitor). These data demonstrate that IS can induce oxidative stress via promoting ROS production in cardiomyocytes, while Klotho has the ability to suppress IS-induced NOX-derived ROS production.It has been reported previously that activations of Mitogen-Activated Protein Kinase (MAPK) pathways, the downstream signals of ROS, play an important role in cardiomyocyte hypertrophy. We therefore explored the relationship between ROS overproduction and MAPK activation in NRCMs treated with IS. Western blot analyses confirmed that the phosphorylations of p38and ERK1/2, but not JNK, were markedly activated in cultured NRCMs after incubation with IS for15-60min. Interestingly, the activations of p38and ERK1/2could be significantly inhibited by pretreatment with10μmol/L DPI or400pmol/L Klotho protein. Moreover, like the effects of Klotho and the inhibitors of p38and ERK1/2(SB203580and U0126), DPI pretreatment significantly inhibited IS-induced increases of3H-leucine uptake and mRNA levels of ANF, BNP and β-MHC. These results indicate that IS-induced cardiomyocyte hypertrophy is probably via activation of ROS/MAPK (p38and ERK1/2) signaling pathways and Klotho can counteract the action of IS.Intraperitoneal injection of Klotho protein ameliorates LVH in uremic miceFinally, we assessed the therapeutic effect of exogenous Klotho protein on CKD-induced LVH in surgically induced renal failure (SIRF) mice. It was shown that Klotho expression in the remaining kidney tissue and its soluble protein in the serum were remarkably decreased at4weeks after nephrectomy. In contrast, the serum IS level was dramatically increased. In comparison with the sham group, the expressions of NOX2and NOX4, as well as the ROS level, were significantly increased in the myocardial tissue in SIRF mice. Notably, treatment of SIRF mice with exogenous Klotho protein (0.01mg/kg/48h) for4weeks led to a significant inhibition of the expressions of NOX2and NOX4, as well as a marked decrease in ROS production, in the myocardial tissue.As expected, a majority of the SIRF mice developed LVH along with the progression of renal failure, while exogenous Klotho protein supplementation resulted in a significant alleviation of LVH, as manifested by a reduction in the relative heart weight, a decrease in ventricular wall thickness, down-regulation of mRNA expressions of cardiac BNP, ANF and β-MHC, as well as decrease in left ventricular internal diameter diastole and increase in left ventricular posterior wall thickness diastole. These results demonstrate that administration of exogenous Klotho protein is effective for the amelioration of CKD-induced LVH.Conclusions:1. IS is an important incentive factor of CKD-induced LVH, which accompanying with a decrease expression of anti-oxidative stress protein named Klotho in kidney. However, the deficiency of Klotho protein could aggravate IS-induced LVH.2. IS contributes to the cardiomyocytes hypertrophy via increasing ROS production by an activation of the NADPH oxidase, which relating to phosphoralation of two down-stream signalling pathways of ROS named p38and ERK1/2. However, the inhibition of expression of NADPH oxidase by ectogenous Klotho protein exerts a vital role in rivalry with IS-induced cardiomyocytes hypertrophy through ROS/MAPKs signalling pathways.3. A significant increase of concentration of serum IS accompanies with a remarkable decrease of Klotho protein in uremia mice modelIn conclusion, our study indicated that uremic toxins played a pivotal role in inducing myocardial hypertrophy and the mechanism was likely to be associated with pro-oxidative stress. As a protein in response to anti-oxidant stress expressed and secreted in the kidney, Klotho can inhibit myocardial hypertrophy induced by uremic toxins. The imbalance between uremic toxins and Klotho is a crucial factor in the development and progression of LVH, while ectogenous Klotho can significantly improve LVH. In this study, we revealed the pathologenesis of uremic myocardiopathy based on the internal correlation between heart and kidney. Simultaneously, we provided a reference for clinical treatment of LVH through up-regulation of endogenous Klotho protein.