Uremia and Inadequate Oxygen Supply Induce Eryptosis and Intracellular Hypoxia in Red Blood Cells.

BACKGROUND/AIMS: Chronic kidney disease is frequently accompanied by anemia, hypoxemia, and hypoxia. It has become clear that the impaired erythropoietin production and altered iron homeostasis are not the sole causes of renal anemia. Eryptosis is a process of red blood cells (RBC) death, like apoptosis of nucleated cells, characterized by Ca2+ influx and phosphatidylserine (PS) exposure to the outer RBC membrane leaflet. Eryptosis can be induced by uremic toxins and occurs before senescence, thus shortening RBC lifespan and aggravating renal anemia. We aimed to assess eryptosis and intracellular oxygen levels of RBC from hemodialysis patients (HD-RBC) and their response to hypoxia, uremia, and uremic toxins uptake inhibition. METHODS: Using flow cytometry, RBC from healthy individuals (CON-RBC) and HD-RBC were subjected to PS (Annexin-V), intracellular Ca2+ (Fluo-3/AM) and intracellular oxygen (Hypoxia Green) measurements, at baseline and after incubation with uremic serum and/or hypoxia (5% O2), with or without ketoprofen. Baseline levels of uremic toxins were quantified in serum and cytosol by high performance liquid chromatography. RESULTS: Here, we show that HD-RBC have less intracellular oxygen and that it is further decreased post-HD. Also, incubation in 5% O2 and uremia triggered eryptosis in vitro by exposing PS. Hypoxia itself increased the PS exposure in HD-RBC and CON-RBC, and the addition of uremic serum aggravated it. Furthermore, inhibition of the organic anion transporter 2 with ketoprofen reverted eryptosis and restored the levels of intracellular oxygen. Cytosolic levels of the uremic toxins pCS and IAA were decreased after dialysis. CONCLUSION: These findings suggest the participation of uremic toxins and hypoxia in the process of eryptosis and intracellular oxygenation.

Uremic toxins promote accumulation of oxidized protein and increased sensitivity to hydrogen peroxide in endothelial cells by impairing the autophagic flux.

Chronic kidney disease (CKD) is associated with high mortality rates, mainly due to cardiovascular diseases (CVD). Uremia has been considered a relevant risk factor for CVD in CKD patients, since uremic toxins (UTs) promote systemic and vascular inflammation, oxidative stress and senescence. Here, we demonstrate that uremic toxins indoxyl sulfate (IxS), p-cresyl sulfate (pCS) and indole acetic acid (IAA) are incorporated by human endothelial cells and inhibit the autophagic flux, demonstrated by cellular p62 accumulation. Moreover, isolated and mixed UTs impair the lysosomal stage of autophagy, as determined by cell imaging of the mRFP-GFP-LC3 protein. Endothelial cells exposed to UTs display accumulation of carbonylated proteins and increased sensitivity to hydrogen peroxide. Rapamycin, an autophagy activator which induces both autophagosome formation and clearance, prevented these effects. Collectively, our findings demonstrate that accumulation of oxidized proteins and enhanced cell sensitivity to hydrogen peroxide are consequences of impaired autophagic flux. These data provide evidence that UTs-induced impaired autophagy may be a novel contributor to endothelial dysfunction.

Low-level arsenic causes proteotoxic stress and not oxidative stress.

Prolonged exposure to arsenic has been shown to increase the risk of developing a number of diseases, including cancer and type II diabetes. Arsenic is present throughout the environment in its inorganic forms, and the level of exposure varies greatly by geographical location. The current recommended maximum level of arsenic exposure by the EPA is 10µg/L, but levels>50-1000µg/L have been detected in some parts of Asia, the Middle East, and the Southwestern United States. One of the most important steps in developing treatment options for arsenic-linked pathologies is to understand the cellular pathways affected by low levels of arsenic. Here, we show that acute exposure to non-lethal, low-level arsenite, an environmentally relevant arsenical, inhibits the autophagy pathway. Furthermore, arsenite-induced autophagy inhibition initiates a transient, but moderate ER stress response. Significantly, low-level arsenite exposure does not exhibit an increase in oxidative stress. These findings indicate that compromised autophagy, and not enhanced oxidative stress occurs early during arsenite exposure, and that restoring the autophagy pathway and proper proteostasis could be a viable option for treating arsenic-linked diseases. As such, our study challenges the existing paradigm that oxidative stress is the main underlying cause of pathologies associated with environmental arsenic exposure.

Plasma cysteine/cystine reduction potential correlates with plasma creatinine levels in chronic kidney disease.

BACKGROUND/AIMS: Oxidative stress has been considered a nontraditional risk factor for cardiovascular disease in the chronic kidney disease (CKD) population, possibly triggered by uremic toxicity. METHODS: A chromatographic method with coulometric detection was adapted to directly and simultaneously determine cysteine (Cys) and cystine (Cyss) in plasma samples. Healthy subjects and CKD subjects in different stages were analyzed. The free Cys and free Cyss levels in their plasma were determined, and the reduction potential [Eh(Cyss/2Cys)] was calculated with the Nernst equation. RESULTS: Healthy plasma presented Eh(Cyss/2Cys) of -123 ± 7 mV. Plasma Eh(Cyss/2Cys) correlated significantly with creatinine levels (p < 0.0001, r = 0.62). CONCLUSION: Plasma Eh(Cyss/2Cys) correlated with increased levels of plasma creatinine, supporting the view that uremia triggers oxidative stress. In addition, it may be used as a quantitative oxidative stress biomarker in uremic conditions.

High-volume hemodiafiltration decreases the pre-dialysis concentrations of indoxyl sulfate and p-cresyl sulfate compared to hemodialysis: a post-hoc analysis from the HDFit randomized controlled trial.

BACKGROUND: Although high-volume online hemodiafiltration has been associated with higher clearance and lower pre-dialysis concentration of middle molecular weight toxins compared to hemodialysis, its effect on protein-bound uremic toxins has shown inconclusive results. In this study, we investigated whether hemodiafiltration impacts pre-dialysis plasma levels of the toxins indoxyl sulfate, p-cresyl sulfate, and indole-3-acetic acid compared to high-flux hemodialysis. METHODS: This is a post-hoc analysis of the multicenter, randomized controlled trial HDFit (ClinicalTrials.gov: NCT02787161). Uremic toxins were determined by high performance liquid chromatography at baseline, 3, and 6 months. Mean differences in monthly changes of pre-dialysis uremic toxin concentrations between hemodiafiltration and high-flux hemodialysis were analyzed using linear mixed-effect models. RESULTS: One hundred ninety-three patients (mean age 53 years old, 71% males) were analyzed. There were no differences between groups regarding clinical and biochemical characteristics at baseline or duration of dialysis session and blood flows throughout the follow-up. Mean differences in rates of change (µM/month, [confidence interval CI]) in high-flux hemodialysis vs. hemodiafiltration were 2.4 [0.3 to 4.56], 3.94 [- 1.54 to 9.41] and 0.06 [- 0.6 to 0.5] for indoxyl sulfate, p-cresyl sulfate and indole-3-acetic acid, respectively. In the exploratory analysis, these differences in high-flux hemodialysis vs. hemodiafiltration subgroup with convective volume > 27.5 L were 2.86 [0.43 to 5.28], 7.43 [0.7 to 14.16] and - 0.19 [- 0.88 to 0.50]. CONCLUSION: These exploratory findings suggest that hemodiafiltration is more effective in reducing indoxyl sulfate as compared to standard high-flux hemodialysis, and also that this effect was extended to p-cresyl sulfate in patients achieving higher convective volumes.

Effect of cranberry supplementation on toxins produced by the gut microbiota in chronic kidney disease patients: A pilot randomized placebo-controlled trial.

BACKGROUND & AIMS: Patients with Chronic Kidney Disease (CKD) have an imbalance in the gut microbiota that can lead to increase levels of lipopolysaccharides (LPS) and uremic toxins such as indoxyl sulfate (IS), p-cresyl sulfate (p-CS), and indole-3 acetic acid (IAA). Among the therapeutic options for modulating gut microbiota are the bioactive compounds such as polyphenols present in cranberry, fruit with potential antioxidant and anti-inflammatory effects. This clinical trial focuses on evaluating the effects of supplementation with a dry extract of cranberry on plasma levels of LPS and uremic toxins in non-dialysis CKD patients. METHODS: It was a randomized, double-blind, placebo-controlled study. Patients were randomized into two groups: the cranberry group received 500 mg of dry cranberry extract (2 times daily), and the placebo group received 500 mg of corn starch (2 times daily) for two months. LPS plasma levels were evaluated by enzyme-linked immunosorbent assay (ELISA) and uremic toxins (IS, p-CS, and IAA) by high-performance liquid chromatography-fluorescence detection. Anthropometric measurements and food intake using the 24-h food recall technique were also evaluated before and after the intervention. RESULTS: Twenty-five participants completed two months of supplementation: 12 patients in the cranberry group (8 women, 56.7 ± 7.5 years, estimated glomerular filtration rate (eGFR) of 39.2 ± 21.9 mL/min); 13 patients in the placebo group (9 women, 58.8 ± 5.1 years, eGFR of 39.7 ± 12.9 mL/min). As expected, there was a negative association between glomerular filtration rate and p-CS and IS plasma levels at the baseline. No change was observed in the uremic toxins and LPS levels. CONCLUSION: Cranberry dry extract supplementation for two months did not reduce the LPS and uremic toxins plasma levels produced by the gut microbiota in non-dialysis CKD patients.

Can curcumin supplementation reduce plasma levels of gut-derived uremic toxins in hemodialysis patients? A pilot randomized, double-blind, controlled study.

BACKGROUND: Gut dysbiosis is common in patients with chronic kidney disease (CKD) and is closely related to inflammatory processes. Some nutritional strategies, such as bioactive compounds present in curcumin, have been proposed as an option to modulate the gut microbiota and decrease the production of uremic toxins such as indoxyl sulfate (IS), p-cresyl sulfate (pCS) and indole-3 acetic acid (IAA). OBJECTIVE: To evaluate the effects of curcumin supplementation on uremic toxins plasma levels produced by gut microbiota in patients with CKD on hemodialysis (HD). METHODS: Randomized, double-blind trial in 28 patients [53.6 ± 13.4 years, fourteen men, BMI 26.7 ± 3.7 kg/m2, dialysis vintage 37.5 (12-193) months]. Fourteen patients were randomly allocated to the curcumin group and received 100 mL of orange juice with 12 g carrot and 2.5 g of turmeric and 14 patients to the control group who received the same juice but without turmeric three times per week after HD sessions for three months. IS, pCS, IAA plasma levels were measured by reverse-phase high-performance liquid chromatography RESULTS: After three months of supplementation, the curcumin group showed a significant decrease in pCS plasma levels [from 32.4 (22.1-45.9) to 25.2 (17.9-37.9) mg/L, p = 0.009], which did not occur in the control group. No statistical difference was observed in IS and IAA levels in both groups. CONCLUSION: The oral supplementation of curcumin for three months seems to reduce p-CS plasma levels in HD patients, suggesting a gut microbiota modulation.

N-acetylcysteine as a potential strategy to attenuate the oxidative stress induced by uremic serum in the vascular system.

AIMS: Chronic kidney disease (CKD) progression is accompanied by systemic oxidative stress, which contributes to an increase in the risk of cardiovascular diseases (CVDs). N-acetylcysteine (NAC) is among the most studied antioxidants, but its therapeutic benefits in CKD-associated CVDs remain controversial. Here, we investigated whether NAC could inhibit the oxidative stress induced by uremia in vitro and in vivo. MAIN METHODS: Endothelial and smooth muscle cells were challenged with human uremic or non-uremic sera, and the effects of a pre-treatment with 2mM NAC were evaluated. Reactive oxygen species (ROS) production, protein oxidation and total glutathione/glutathione disulfide (tGSH/GSSG) ratios were measured. Five-sixths nephrectomized or sham-operated rats were orally treated (in the drinking water) with 60 mg/kg/day NAC or not treated for 53 days. Plasma cysteine/cystine reduction potential Eh(Cyss/2Cys) was determined as a novel marker of the systemic oxidative stress. KEY FINDINGS: NAC inhibited all the determined oxidative stress parameters, likely by increasing the tGSH/GSSG ratio, in both cell lines exposed to uremic serum. Orally administered NAC attenuated the systemic oxidative stress in uremic rats. SIGNIFICANCE: The present results indicate that NAC, by preventing GSH depletion in vascular cells exposed to uremic serum and by attenuating the systemic oxidative stress during CKD progression, emerges as a potential strategy to prevent the oxidative stress induced by uremic toxicity in the vascular system.