Presentation Summary

Written by Miljan Krcobic
Reviewed by Leticia Prates Roma

Patients with chronic kidney disease (CKD) carry a 5- to 10-fold higher risk for developing cardiovascular disease (CVD) compared to age-matched controls [1]. Heart failure (HF) is the leading CV complication in CKD patients and its prevalence increases with declining kidney function. Approximately 50% of patients with CKD stages 4 and 5 will develop HF [2]. Although the exact mechanism leading to such events is not known, reactive oxygen species (ROS) are considered as one of the potential mediators [1].

The main sources of ROS production in CKD and visualization techniques

Growing evidence supports oxidative injury as a common link between progressive renal and cardiac dysfunction. Because both primary cardiac and renal failures lead to the elaboration of the renin-angiotensin-aldosterone system (RAAS), activation of oxidases by angiotensin II in one organ has the potential to lead to progressive dysfunction in the secondary organ through ROS generation (Figure 1). It has also been suggested that volume overload and cardiac preload and afterload caused by RAAS activation may lead to a mitochondrial imbalance in cardiomyocytes, further increasing the ROS production and potentially initiating HF in CKD patients [3].

Figure 1: Classical concept of ROS production in CKD (Slide 5 [4]).

ROS are mainly produced by mitochondria, NADPH oxidases (NOX), and other enzymes like xanthine oxidase and cytochrome P450. To be able to differentiate the production of ROS between mitochondria and NOX, it is necessary to measure ROS and detect their specific changes in different cell-like compartments [5]. However, a lack of tools for measuring the status of defined intracellular redox couples has been a profound limitation for many years. Conventional approaches either lacked well-defined specificity or disrupted cellular integrity. For example, redox-sensitive fluorescent dyes, frequently used to detect oxidant generation within cells, interact with multiple oxidants and may even promote artificial ROS formation [6]. To overcome these limitations, new, genetically encoded biosensors such as redox-sensitive green fluorescent protein (roGFP2) coupled with human glutaredoxin-1 (Grx1) (Grx1-roGFP2) and oxidant receptor peroxidase 1 (Orp1)-roGFP2 were developed [6, 7]. The advantages of these sensors comprise higher specificity and sensitivity for particular ROS, better targeting to different cell line compartments including even microenvironment inside the cell and the possibility to make ratiometric imaging and real-time measurements [8].

Recently, Prof Roma and her team developed a procedure that enables the preservation and in vivo visualization of the redox state of an expressed biosensor (mitochondrial Grx1-roGFP2 or Orp1-roGFP2) in transgenic mice. This technique allowed visualization of redox changes such as, for example, H2O2 production in the mitochondria, that may occur during embryonic development of mice (yellow spots on Figure 2A) or the necrotic core oxidation in mice with small cells lung cancer (Figure 2B) [9]. Another advantage of this procedure is that different cell-types including cardiomyocytes of mice can be isolated and ex vivo dynamic measurements can be performed [4].

Figure 2A. Visualization of the oxidation in a mouse embryo; Figure 2B. Visualization of the oxidation in small cells in a mouse with lung cancer (Slide 5 [4]) [9].

Additionally, one study investigated whether mitochondria and cytosolic ROS are produced in the heart of CKD mice using a novel method for induction of renal failure through dietary delivery of adenine. After 11 weeks of follow-up, mice developed only mild cardiac phenotype followed by increased plasma creatinine levels, an increase in systolic blood pressure, early cardiac volumetric parameter impairment and increased mitochondrial oxidation [10, 4].

Uremic toxins as mediators of cardiac failure

Uremic toxins may also play an important role in the progression of CV disease in the setting of CKD. The highly protein-bound uremic toxin indoxyl sulfate has emerged as a potent toxin adversely affecting both the kidney and heart. Direct cardiac effects of this and other uremic toxins have been recently demonstrated in in vitro and in vivo studiesPotent fibrogenic and prohypertrophic effects, as well as oxidative stress-inducing effects, appear to play a central role in uremic-toxin driven renal and cardiac pathology [11].

In one of the experiments, redox sensors were used to screen whether different uremic toxins can be involved in cardiac failure. Researchers stably expressed H2O2 biosensors in both the cytosol and mitochondria of the cardiomyocytes. The sensors proved to be very responsive to H2O2 levels. In mitochondria, H2O2 concentration of 5 μM already led to mitochondria response, whereas in the cytosol, the response was observed when the concentrations of H2O2 were 20 μM or higher. Sensors were saturated when the levels of H2O2 were between 50 and 100 μM. Results of this analysis confirmed increased cytosolic and mitochondrial H2O2 levels with fractions from the dialysate, and increased cell death in the hydrophilic fraction partially protected by protein SS-31 [4].


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