Supposed mechanism of PGC1-α/NRF1-NRF2 pathway anti-oxidative stress cellular defense in chronic kidney disease patients in peritoneal dialysis treatment.

<p>Oxidative stress alters the interaction of Kelch-like ECH-associated protein 1 (Keap1) and Nuclear factor erythroid-derived 2-like 2 (Nrf2), thereby liberating Nrf2 activity from repression by Keap1. NRF2 migrates into the nucleus were it activates the transcription of Superoxide dismutase 2, mitochondrial (SOD2). At the same time, oxidative stress causes the down-regulation of Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1-α) and Nuclear respiratory factor-1 (NRF-1) with the consequent down-regulation of PGC-1α downstream target genes (TFAM, COX6C, COX7C, UQCRH and MCAD). The reduced TFAM expression causes a decrease in mitochondrial transcription and replication. The down-regulation of all these factors suggests the decrease in mitochondrial OXPHOS activity in order to reduce ROS accumulation and creating an antioxidant feedback.</p

Demographics and clinical characteristics of study group.

<p>Values are expressed as mean ± SD. GN, glomerulonephritis; ADPKD, adult dominant polycystic disease; RVD, renal vascular disease; ESRD, end-stage renal disease. BMI, body mass index; Hs-CRP, high sensitivity- C reactive protein. p-values determined by t-test and Chi-squared test.</p

Resting metabolic rate (RMR) assessment in healthy subjects (HS) and chronic kidney disease patients in peritoneal dialysis (PD).

<p>Dot-plot represents the RMR assessed by indirect calorimetry in 15 HS and 15 PD patients. RMR levels were similar in the two study groups.</p

COX6C, COCX7C and UQCRH gene expression by Real-Time PCR in PBMCs from healthy subjects (HS) and chronic kidney disease patients in peritoneal dialysis (PD).

<p>The histograms represent the mean ± SD of COX6C (A) COX7C (B) and UQCRH (C) expression level, determined by Real-Time PCR in PBMCs from 15 HS and 15 PD patients. For all these genes the expression level was significantly lower in PD compared to HS (**p<0.001).</p

Transcription factor A, mitochondrial (TFAM) gene and protein expression in PBMCs from healthy subjects (HS) and chronic kidney disease patients in peritoneal dialysis (PD).

<p>(A) The histogram represents the mean ± SD of TFAM expression level, determined by Real-Time PCR in PBMCs from 15 HS and 15 PD patients. (B) Dot-plot represents the normalized TFAM protein level in total cell lysates of 8 HS and 11 PD patients assessed by Western blotting and (C) the representative western blotting experiment for TFAM. The line represents the mean value. Both TFAM mRNA and protein level was significantly lower in PD patients compared to HS.</p

Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1-α) and nuclear respiratory factor-1 (NRF-1) gene expression by Real-Time PCR in PBMCs from healthy subjects (HS) and chronic kidney disease patients in peritoneal dialysis (PD).

<p>The histograms represent the mean ± SD of PGC1-α (A) and NRF-1 (B) expression level, determined by Real-Time PCR in PBMCs from 15 HS and 15 PD patients. For both genes the expression level was significantly lower in PD compared to HS (**p<0.001).</p

Nuclear factor erythroid-derived 2-like 2 (NRF2) protein expression in nuclear extracts of PBMCs from healthy subjects (HS) and chronic kidney disease patients in peritoneal dialysis (PD).

<p>(A) Dot-plot represents the normalized NRF2 protein level in nuclear extracts of 5 HS and 5 PD patients assessed by Western blotting and (B) the representative western blotting experiment for NRF2. The line represents the mean value. NRF2 protein level was significantly higher in PD patients compared to HS.</p

Mitochondrial ROS production by FACS in PBMC from healthy subjects (NORM) and hemodialyzed chronic kidney disease patients (CKD-HD).

<p>Freshly isolated PBMC from 10 NORM and 10 CKD were incubated with 5 μM Mitosox RED for 10 min at 37°. After centrifugation at 4°C the cells were analyzed for Mitosox signal in a flow cytometer. (A and B) Representative FACS image of PBMC from one NORM and one CKD-HD patient, respectively. (C) Histogram indicates median value ± SD of Mitosox positive cells in CKD-HD and NORM. CKD-HD patients showed higher number of positive cells compared to NORM (*p<0.05).</p

NOD-like receptor 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD domain (ASC), caspase-1 (CASP-1), IL-1β, IL-18 and P2XR7 gene expression by Real-Time PCR in PBMC from healthy subjects (NORM) and hemodialyzed chronic kidney disease patients (CKD-HD).

<p>Histograms represent the mean ± SD of (A) NLRP3, (B) ASC, (C) CASP-1, (D) IL-18, (E) IL-1β and (F) P2XR7 mRNA levels determined by Real-Time PCR in PBMC isolated from 15 NORM and 15 CKD-HD patients. For all genes, expression levels resulted significantly higher in CKD-HD compared to NORM (*p<0.05, **p<0.01).</p

pCS-IS and total cholesterol-FMD correlations.

<p>Graphical representation of the direct correlation between pCS and IS (Fig 5a) and of the inverse correlation between total cholesterol and FMD (Fig 5b). Round and squared points represents T0 and T2 values, respectively. Correlation coefficients and p values are represented in the figure.</p