In the last twenty years, several reports have focused on the molecular changes occurring during the onset and progression of CKD, but this rich literature appears fragmented and not exhaustive [10
]. Analyzing these reports the attitude to focus on small number of biological elements and the lack of a comprehensive strategy to study the biochemical network associated with CKD is evident. In addition, despite the efforts of researchers and clinicians, CKD and renal replacement therapy are still associated to important clinical complications. In particular, during HD, the interaction of PBMC with dialytic membranes causes their activation with a consequent increased synthesis and release of pro-inflammatory cytokines [9
] and imbalance between pro- and anti-oxidant activities, resulting in high oxidative stress with elevated synthesis of ROS [13
In the present study, we used a whole genome analysis by microarray technology, combined with classical biomolecular approaches, to detect unrecognized biological elements deregulated in subjects with CKD and to identify new potential targets for pharmacological interventions. Microarray analysis revealed a specific genomic fingerprint able to identify HD and CKD patients from healthy subjects. Functional analysis by IPA and KEGG demonstrated that 25% of the selected genes encodes for protein involved in mitochondrial oxidative phosphorylation system.
Mitochondria are essential eukaryotic cells organelles involved in several metabolic pathways, calcium and iron homeostasis, ROS production and programmed cell death [28
]. They present an outer and inner membrane, the latter of which would be impermeable to all molecules in the absence of specific carriers and contains the enzymatic oxidative phosphorylation complex. The respiratory flux is due to the donation of electrons from NAD- or FAD-dependent substrates, via respiratory chain, to molecular oxygen which is finally reduced to water. Simultaneously, the energy conserving complexes I, III and IV build up a trans-membrane electrochemical gradient by coupling the electron transfer activity to proton translocation from the matrix to the outer side of the inner mitochondrial membrane. Complex V utilizes backward the electrochemical gradient for ATP synthesis.
A significant number of the genes discriminating CKD and HD patients from healthy subjects were involved in the synthesis of important nuclear-encoded structural subunits of the oxidative phosphorylation complexes. In particular, NDUFA6
B1 encode for subunits of the Complex I (NADH dehydrogenase) that is involved in the transfer of electrons from NADH to ubiquinone [29
]. Interestingly, another gene encoding for a subunit of this complex (NDUFA2
) was identified by microarray analysis being up-regulated in skeletal muscle biopsy specimens of HD patients compared to control subjects without renal failure [27
encode for two subunits of the cytochrome c oxidase (COX or Complex IV), the terminal enzyme of the mitochondrial respiratory chain catalyzing the electron transfer from reduced cytochrome c to oxygen [30
encode for components of the ATP synthase (complex V).
To investigate whether the increased gene expression observed in our genomic study was, indeed, associated with an increased oxidative phosphorylation system activity, we analyzed the protein expression of mitochondrial-coding COXI (catalytic subunit) and nuclear-coding COXIV subunits of Complex IV. Although these two essential subunits of Complex IV have not been identified by our genomic approach, we decided to measure their levels based on the evidence that COX exerts a tight control on the respiration of a variety of human cells including myeloma [31
] and Jurkat blood cells [32
]. The observation that the expression of both proteins was higher in HD and CKD patients compared to healthy subjects further confirmed the data obtained by the microarray analysis.
Additionally, when we measured the activity of complex IV, despite the expected high interindividual variability [33
], we observed a dramatic reduction in both CKD and HD patients compared to healthy subjects. This is in line with experimental evidences suggesting that chronic oxidative stress and oxidant injury may enhance the expression of several nuclear mitochondrial biogenesis genes [34
]. Indeed, our results highlight the role of oxidative stress in this process since both CKD and HD patients were characterized by an elevated intracellular ROS production and a high level of 8-OHdG, a marker of oxidative stress to DNA [36
]. ROS may deeply influence a variety of key cell functions damaging proteins, lipids and nucleic acids [37
] and inhibiting directly the enzymatic activities of several elements of the cellular respiratory chains [40
]. Thus, our hypothesis is that an increased production of ROS due to the effect of pro-inflammatory mediators may cause a profound inhibition of the oxidative phosphorylation system leading to a compensatory "hypertrophy" of its components. In addition, a hypertrophic and impaired oxidative phosphorylation system may prime a vicious circle, causing a continuous release of ROS.
An interesting point in our study is that CKD and HD patients are virtually undistinguishable when it comes to the expression of oxidative phosphorylation system components. On this basis, we may consider this finding as a genomic hallmark of CKD itself that is not modulated by HD treatment. However, it should be taken into consideration that our HD patient's population has been treated with highly biocompatible synthetic membranes, previously shown to cause a very limited lymphomononuclear cell activation [43
], although, Raj DS et al. have reported that mitochondrial dysfunction is induced even with the use of biocompatible membrane [46