Heme oxygenase-1 and chronic kidney disease: A translational study of oxidative stress and atherogenesis

In advanced kidney disease the prevalence of atherosclerotic cardiovascular disease is high and nearly equivalent across age groups. Recent data indicate that even minor renal dysfunction impacts on the onset of cardiovascular disease (CVD). Many authors hypothesize that increased oxidative stress is the unifying concept that initiates and feeds this problem. Several findings in uremic patients point towards an imbalance favoring the pro-oxidative state: uremia-related as well as dialysis-related factors have been described to lead to the increase of oxidants and decrease of the antioxidative capacity.  

In Chapter 1 we summarized current knowledge on pathophysiological mechanisms of oxidative stress, antioxidant defense mechanisms and atherosclerotic vascular disease in uremia. Diverse mechanisms, both endogenous as exogenous, lead to the increased activity of oxidative enzymes, the accumulation of secondary radicals and/or transition metals and the impairment of crucial antioxidative mechanisms.

The overall aim of this thesis was to explore the anti-oxidative and anti-atherosclerotic enzyme Heme oxygenase-1 (HO-1) in the context of uremia and accelerated atherosclerosis (Chapter 2).

In Chapter 3 we report on our evaluation of atherosclerotic plaque morphology and HO-1 expression in patients with known advanced atherosclerotic disease of the peripheral arterial vasculature. In 66 biopsies of arteries procured during clinically indicated vascular surgery procedures, atherosclerotic lesions in CKD patients were characterized by higher inflammatory cell infiltration, more neovascularization and a higher proportion of plaque complications than seen in lesions of patients without renal insufficiency. In agreement, the degree of renal impairment (eGFR) was found to be an important predictor of both intra-plaque neovascularization and atherosclerotic plaque complications, independent of traditional cardiovascular risk factors. Atherosclerotic lesions in CKD patients had a tendency to lower expression of the anti-atherogenic enzyme HO-1.

To further explore the effect of the uremic environment on HO-1 expression in the early phase of atherogenesis, we conducted an in vitro assay in human umbilical arterial endothelial cells (HUAECs) preconditioned with either healthy or uremic human serum, as described in Chapter 4. Moreover, we studied the effect of HO-1 overexpression on oxidative stress-induced cell injury in both healthy and uremic conditions. We found that HUAECs show a higher vulnerability to the oxidative stressor ONOO- after preconditioning in uremic serum as compared to healthy serum. This could not be explained by differential effects of the culture conditions on the expression of the protective enzyme HO-1 or reactive oxygen species (ROS) generation. The exact mechanism behind the increased vulnerability remains to be unraveled. Interestingly however, Hemin-induced HO-1 overexpression mitigated this cell vulnerability in uremic conditions by normalizing the apoptotic tendency and by reducing cytoplasmic ROS generation.

In Chapter 5 two studies on the impact of known functional HO-1 promoter polymorphisms on the occurrence of CVD are reported. In Chapter 5.1, we describe the results of a systematic review of the literature on the role of the HO-1 (GT)n repeat polymorphism in the development of atherosclerotic disease in the general population. Shorter repeats with (GT)n<25 are associated with higher inducibility and activity of HO-1. The epidemiological association between HO-1 (GT)n promoter polymorphism and CVD seen in our review, confirmed the presumed protective effects of HO-1 enzymatic activity. In addition, variability in genotype distribution between ethnic groups was shown, which should be taken into account when interpreting data from published studies.

In Chapter 5.2 we studied the role of the HO-1 (GT)n repeat polymorphism and the HO-1 T(-413)A Single Nucleotide Polymorphism (SNP) in the occurrence of CVD in a dialysis population. Like shorter (GT)n repeats, the A allele of the SNP is associated with higher inducibility and activity of HO-1. Our study provided evidence for a role of the (GT)n repeat promoter polymorphism of the HO-1 enzyme in the occurrence of CVD in a dialysis population. Dialysis patients with a biallelic long genotype (lower HO-1 expression) had an increased risk for CVD compared to patients with biallelic short genotype (higher HO-1 expression). On the other hand we could not show any significant association between the T(-413)A SNP and CVD.

From a detailed discussion of our experimental data (Chapter 6), we conclude that the present project adds to the current understanding of the accelerated atherosclerosis process seen in CKD. From our findings, it is clear that the uremic environment increases the vulnerability to oxidative stressors of the vasculature as a whole as well as of the endothelial cells in particular. Heme Oxygenase-1 was studied in many different conditions and its expression appears not to be impacted by CKD at first sight. However, taking the stress-responsive nature of the enzyme into account, the findings of both our clinical and in vitro studies may suggest relative deficiency of the HO-1 defense mechanism in uremia. Irrespective of the above interpretation, we found that known functional HO-1 promoter polymorphisms play a similar role in CVD in CKD patients as they do in the general population. Furthermore an interventional strategy, consisting of strong selective induction of HO-1 was capable of restoring the uremia-induced cell vulnerability. Given the pluripotency of HO-1 with its many anti-inflammatory and diverse anti-oxidative effects we believe that HO-1 constitutes an attractive and promising target molecule for further interventional research. The exact protective mechanism of HO-1 overexpression in this setting needs further elucidation.