Aportes de la biotecnología al diagnóstico y tratamiento de la enfermedad renal crónica y comorbilidades frecuentes / Biotechnology contribution to the diagnosis and treatment of chronic kidney disease and frequent comorbidities

Este trabajo tiene como objetivo revisar las contribuciones de la biotecnología, en relación con el tratamiento, diagnóstico y la monitorización de la enfermedad renal crónica (ERC) y sus comorbilidades más frecuentes, especialmente la anemia. En relación con los tratamientos, enfocamos el desarrollo de productos biofarmacéuticos como los agentes estimulantes de la eritropoyesis (ESA), que fueron los primeros biofármacos utilizados para el tratamiento de la anemia asociada a la ERC; analizamos sus características y utilización actual después de varios años de experiencia clínica, así como también otras alternativas en desarrollo. Revisamos distintos tipos de bioterapias, la utilización de las células estromales mesenquimales de médula ósea (MSC) y tratamientos alternativos con modificaciones dietarias, que se basan en la asociación entre la microbiota intestinal de los pacientes renales crónicos y sus condiciones fisiopatológicas. Finalmente, en relación con el diagnóstico y monitorización, nos referimos al estudio y validación de biomarcadores diagnósticos, predictivos y terapéuticos que han permitido optimizar los resultados clínicos en este tipo de pacientes. (AU)

The aim of this work is to review the contributions of biotechnology, in relation to the treatment, diagnosis and monitoring of chronic kidney disease (CKD) and its most frequent comorbidities, especially anemia. Regarding the treatment, we focus on the development of biopharmaceutical products such as erythropoiesis stimulating agents (ESA), which were the first biopharmaceuticals used to treat anemia associated with chronic kidney disease (CKD). We analyzed their characteristics and their current use after several years of clinical experience, as well as other alternatives in development. We also review different types of biotherapies, the use of bone marrow mesenchymal stromal cells (MSC) and alternative treatments with dietary modifications, which are based on the association between the intestinal microbiota of chronic kidney patients and their pathophysiological conditions. Finally, in relation to diagnosis and monitoring, we refer to the study and validation of diagnostic, predictive and therapeutic biomarkers that have made clinical results possible to be optimized in this type of patient. (AU)

Acai Extract Transiently Upregulates Erythropoietin by Inducing a Renal Hypoxic Condition in Mice.

Acai (Euterpe oleracea Mart. Palmae, Arecaceae) is a palm plant native to the Brazilian Amazon. It contains many nutrients, such as polyphenols, iron, vitamin E, and unsaturated fatty acids, so in recent years, many of the antioxidant and anti-inflammatory effects of acai have been reported. However, the effects of acai on hematopoiesis have not been investigated yet. In the present study, we administered acai extract to mice and evaluated its hematopoietic effects. Acai treatment significantly increased the erythrocytes, hemoglobin, and hematocrit contents compared to controls for four days. Then, we examined the hematopoietic-related markers following a single injection. Acai administration significantly increased the levels of the hematopoietic-related hormone erythropoietin in blood compared to controls and also transiently upregulated the gene expression of Epo in the kidney. Furthermore, in the mice treated with acai extract, the kidneys were positively stained with the hypoxic probe pimonidazole in comparison to the controls. These results demonstrated that acai increases the erythropoietin expression via hypoxic action in the kidney. Acai can be expected to improve motility through hematopoiesis.

Laboratory evidence for the hematopoietic potential of Beta vulgaris leaf and stalk extract in a phenylhydrazine model of anemia.

This study was designed to provide laboratory evidence supporting the hematopoietic effect of Beta vulgaris (beet) leaf aqueous extract in phenylhydrazine-induced anemia model in albino rats. Extraction of the leaves/stalks was done by maceration in 30% hydro-ethanol for 48 h. An intraperitoneal injection of 20 mg/kg phenylhydrazine was applied for two consecutive days to develop hemolytic anemia on the 4th day after the 1st injection in 24 of 30 male albino rats. The animals were divided into 5 groups and received the following treatments: standard (ferrous ascorbate + folic acid; 13.5 + 0.135 mg/kg), B. vulgaris extract (100 and 200 mg/kg), or left untreated (normal and diseased controls). Blood samples were taken at 0, 4, 8, and 12 days of the experiment for hematological and clinico-chemical analysis. Beet leaf extract significantly restored the levels of red blood cells, white blood cells, hemoglobin, and hematocrit in dose- and time-dependent manners. Blood indices have been significantly corrected. Erythropoietin level was maintained at higher levels. Erythrocytic membrane oxidation biomarker (malondialdehyde) level was significantly reduced compared to the anemic untreated group. The extract exhibited potent, concentration (4-512 µg/mL)-dependent antioxidant activity indicated by the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay, with IC50 value of 37.91 µg/mL. Beet leaf extract resulted in detection of flavonoid and phenolic compounds that may underlie its hematinic properties. These findings may indicate B. vulgaris as a good natural source for pharmaceutical preparations with hematopoietic effects and treatment of anemia and/or associated conditions.

Laboratory evidence for the hematopoietic potential of Beta vulgaris leaf and stalk extract in a phenylhydrazine model of anemia

This study was designed to provide laboratory evidence supporting the hematopoietic effect of Beta vulgaris (beet) leaf aqueous extract in phenylhydrazine-induced anemia model in albino rats. Extraction of the leaves/stalks was done by maceration in 30% hydro-ethanol for 48 h. An intraperitoneal injection of 20 mg/kg phenylhydrazine was applied for two consecutive days to develop hemolytic anemia on the 4th day after the 1st injection in 24 of 30 male albino rats. The animals were divided into 5 groups and received the following treatments: standard (ferrous ascorbate + folic acid; 13.5 + 0.135 mg/kg), B. vulgaris extract (100 and 200 mg/kg), or left untreated (normal and diseased controls). Blood samples were taken at 0, 4, 8, and 12 days of the experiment for hematological and clinico-chemical analysis. Beet leaf extract significantly restored the levels of red blood cells, white blood cells, hemoglobin, and hematocrit in dose- and time-dependent manners. Blood indices have been significantly corrected. Erythropoietin level was maintained at higher levels. Erythrocytic membrane oxidation biomarker (malondialdehyde) level was significantly reduced compared to the anemic untreated group. The extract exhibited potent, concentration (4-512 μg/mL)-dependent antioxidant activity indicated by the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay, with IC50 value of 37.91 μg/mL. Beet leaf extract resulted in detection of flavonoid and phenolic compounds that may underlie its hematinic properties. These findings may indicate B. vulgaris as a good natural source for pharmaceutical preparations with hematopoietic effects and treatment of anemia and/or associated conditions.

Impact of Erythropoiesis-Stimulating Agents on Behavioral Measures in Children Born Preterm.

OBJECTIVE: To evaluate the impact of erythropoiesis-stimulating agents (ESAs) administered during initial hospitalization and family demographic factors on behavior at 3.5-4 years of age. STUDY DESIGN: Children were enrolled who had previously participated in a randomized study of ESAs (n = 35) or placebo (n = 14) in infants born preterm with birth weights of 500-1250 g. A term healthy control group (n = 22) also was recruited. Behavior was evaluated by parent report with the Behavioral Assessment System of Children-2. Principal component analyses identified 2 demographic factors, a Socioeconomic Composite (SEC) and a Family Stress Composite. A multivariate general linear model evaluated the impact of study group and sex on the 4 composite scales of the Behavioral Assessment System of Children-2. Demographic factors were treated as covariates and interactions with study group (ESA, placebo, and term) were examined. RESULTS: The ESA group had significantly better scores than the placebo group on behavioral symptoms (P = .04) and externalizing scales (P = .04). An interaction was observed between study group and SEC (P = .001). A beneficial effect of ESAs was maximal in the children with lower SEC scores. CONCLUSIONS: The beneficial effects of ESAs on childhood behavior were maximal in children with lower SEC scores. ESAs seemed to ameliorate the adverse impact of lower SEC on behavioral domains seen in the placebo group. This effect was independent of the beneficial effect of ESAs on global cognition we reported previously. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01207778 and NCT00334737.

Iron dextran increases hepatic oxidative stress and alters expression of genes related to lipid metabolism contributing to hyperlipidaemia in murine model.

The objective of this study was to investigate the effects of iron dextran on lipid metabolism and to determine the involvement of oxidative stress. Fischer rats were divided into two groups: the standard group (S), which was fed the AIN-93M diet, and the standard plus iron group (SI), which was fed the same diet but also received iron dextran injections. Serum cholesterol and triacylglycerol levels were higher in the SI group than in the S group. Iron dextran was associated with decreased mRNA levels of pparα, and its downstream gene cpt1a, which is involved in lipid oxidation. Iron dextran also increased mRNA levels of apoB-100, MTP, and L-FABP indicating alterations in lipid secretion. Carbonyl protein and TBARS were consistently higher in the liver of the iron-treated rats. Moreover, a significant positive correlation was found between oxidative stress products, lfabp expression, and iron stores. In addition, a negative correlation was found between pparα expression, TBARS, carbonyl protein, and iron stores. In conclusion, our results suggest that the increase observed in the transport of lipids in the bloodstream and the decreased fatty acid oxidation in rats, which was promoted by iron dextran, might be attributed to increased oxidative stress.

Acetylsalicylic acid mitigates erythropoietin-associated blood pressure increase in nonuremic rats.

BACKGROUND: Approximately 30% of the chronic kidney disease patients using recombinant human erythropoietin (rhuEPO) have an increase in blood pressure (BP). Its mechanism and whether it depends on renal function remain unclear. There is early evidence that acetylsalicylic acid (ASA) prevents the rhuEPO-induced increase in BP. This study aims to verify whether very high doses of rhuEPO can increase BP in nonuremic rats and whether the co-administration of ASA can prevent it. METHODS: Forty male Wistar rats were divided into four groups: placebo/placebo; placebo/rhuEPO 200 UI/kg thrice weekly; placebo/ASA 50 mg/kg daily; rhuEPO 200 UI/kg thrice weekly/ASA 50 mg/kg daily. Hematocrit was measured before and after and systolic BP was measured weekly by tail-cuff technique. Direct measurement of the BP was obtained at the end. RESULTS: The rhuEPO groups had higher final hematocrit (rhuEPO/placebo 56.7 ± 7.6, rhuEPO/ASA 56.7 ± 7.7; p < 0.001 versus placebo/placebo, 42.2 ± 4.7 and ASA/placebo 41.2 ± 4.2); and also increase in systolic BP (rhuEPO/placebo 135.1 ± 15.0, p = 0.01 and rhuEPO/ASA 127.2 ± 6.8, p = 0.02), whereas BP in rats from placebo/placebo (120.9 ± 5.0, p = 0.18) and placebo/ASA (124.6 ± 13.3, p = 0.12) groups remained unchanged. By direct measurement, the final BP was higher in rhuEPO/placebo (DBP 123.1 ± 12.0; SBP 157.4 ± 12.5; MBP 139.8 ± 11.9) than placebo/placebo (DBP 105.1 ± 11.5; SBP 141.0 ± 12.6; MBP 122.1 ± 12.1) and placebo/ASA groups (DBP 106.6 ± 8.1; SBP 141.5 ± 8.4, MBP 122.1 ± 7.2) (p < 0.05 by post hoc Bonferroni test ANOVA). The rhuEPO/ASA group (PAD 115.1 ± 11.4, PAS 147.4 ± 9.1, MBP 130.1 ± 10.3) was not different from other groups. CONCLUSIONS: The administration of very high doses of rhuEPO is associated with an increase in hematocrit and BP in nonuremic rats. The concomitant use of ASA mitigates the rhuEPO-associated BP increase.

Effects of iron deficiency anemia and its treatment with iron polymaltose complex in pregnant rats, their fetuses and placentas: oxidative stress markers and pregnancy outcome.

OBJECTIVE: Iron deficiency anemia (IDA) can severely impair the outcome of pregnancy. IDA has been shown to cause oxidative stress, which may be exacerbated by oral iron therapy. In this study, the effects of IDA and its treatment with iron polymaltose complex/folic acid (IPC/FA) were examined in anemic pregnant rats, their fetuses and placentas. STUDY DESIGN: Hematological variables and oxidative stress markers in the liver, heart and kidney were evaluated in non-anemic, anemic and IPC/FA-treated pregnant rats and their fetuses. Markers for oxidative stress, inflammation and hypoxia were assessed in the placentas of all groups. RESULTS: IDA was shown to increase oxidative stress levels in all the studied organs and in placenta as well as hypoxia and inflammation in placenta. IPC/FA treatment corrected IDA measured by the hemoglobin level, serum iron level and transferrin saturation. The oxidative stress levels in all the studied organs and in placentas of the IPC/FA-treated group were comparable to those of the non-anemic group. The number of fetuses and the neonatal and placental weight were lower in the anemic group compared to the non-anemic and IPC/FA-treated groups. CONCLUSIONS: The current study shows that IDA in pregnant rats impaired pregnancy outcome, increased the expression of hypoxia and inflammatory markers in the placenta, and increased oxidative stress in dams, fetuses and placentas. Treatment with oral IPC/FA corrected the IDA as well as reduced the levels of oxidative stress and inflammatory markers close to non-anemic control values in all the studied organs.

Labile plasma iron generation after intravenous iron is time-dependent and transitory in patients undergoing chronic hemodialysis.

Iron supplementation in hemodialysis patients is fundamental to erythropoiesis, but may cause harmful effects. We measured oxidative stress using labile plasma iron (LPI) after parenteral iron replacement in chronic hemodialysis patients. Intravenous iron saccharate (100 mg) was administered in patients undergoing chronic hemodialysis (N = 20). LPI was measured by an oxidant-sensitive fluorescent probe at the beginning of dialysis session (T0), at 10 min (T1), 20 min (T2), and 30 min (T3) after the infusion of iron and at the subsequent session; P < 0.05 was significant. The LPI values were significantly raised according to the time of administration and were transitory: -0.02 +/- 0.20 micromol/L at the beginning of the first session, 0.01 +/- 0.26 micromol/L at T0, 0.03 +/- 0.23 micromol/L at T1, 0.09 +/- 0.28 micromol/L at T2, 0.18 +/- 0.52 micromol/L at T3, and -0.02 +/- 0.16 micromol/L (P = 0.001 to 0.041) at the beginning of the second session. The LPI level in patients without iron supplementation was -0.06 +/- 0.16 micromol/L. Correlations of LPI according to time were T1, T2, and T3 vs. serum iron (P = 0.01, P = 0.007, and P = 0.0025, respectively), and T2 and T3 vs. transferrin saturation (P = 0.001 and P = 0.0003, respectively). LPI generation after intravenous saccharate administration is time-dependent and transitorily detected during hemodialysis. The LPI increment had a positive correlation to iron and transferrin saturation.

Differences between original intravenous iron sucrose and iron sucrose similar preparations.

Iron sucrose (Venofer; reference) has a good safety record and is prescribed in patients with anaemia and chronic kidney disease worldwide, but various iron sucrose similar (ISS) preparations are now utilized in clinical practice. This study evaluates possible differences between iron sucrose and ISS preparations on haemodynamic and oxidative stress markers in normal rats. 60 male and 60 female Sprague Dawley rats were divided into four groups and assigned to receive commercially available ISS test 1, ISS test 2, reference or isotonic saline solution (control). A single i.v. dose of iron (40 mg/kg) or saline (equivalent volume) was administered after 24 h and every 7 days for 4 weeks. Blood samples were collected for biological assessment of haemoglobin (Hb), serum iron and percentage transferrin saturation (TSAT), and urine samples were collected to investigate creatinine clearance and proteinuria. Animals were sacrificed after receiving an i.v. dose on days 1, 7 and 28, and kidney, liver, and heart homogenates were then collected to determine antioxidant enzyme levels. Tissues were processed using Prussian blue and immmunohistochemistry techniques to identify iron deposits, tissue ferritin and pro-inflammatory markers. Systolic blood pressure was significantly reduced in the ISS groups relative to the reference and control groups after 24 h and on days 7, 14 and 21 (p < 0.05). Creatinine clearance was reduced (p < 0.01) and proteinuria marked (p < 0.01) in the ISS groups at 24 h and on days 7 and 28 relative to the reference and control groups which did not differ throughout the study. Liver enzymes were also increased in the ISS groups at 24 h and on days 7 and 28. Both ISS test 1 and ISS test 2 groups presented a significant increase in catalase, thiobarbituric reactive species, Cu, Zn-superoxide dismutase (CuZnSOD) and glutathione peroxidase activity, and a decrease in glutathione levels (p < 0.01) in the liver, heart and kidney at 24 h and on day 7 relative to the reference and control groups. Serum iron and percentage TSAT were elevated in all groups (except control) (p < 0.01) but no differences in Hb concentration were observed between them. Finally, levels of the proinflammatory markers TNF-alpha and IL6 were significantly elevated in the ISS groups (liver, heart and kidney) compared with the reference and control groups on day 28 (p < 0.01). These findings suggest significant differences between the reference and ISS test 1/ISS test 2 regarding oxidative stress and the inflammatory responses of liver, heart and kidneys in normal rats. A possible explanation for these observations could be the stability of the iron complex.

Effect of hydroquinone, hydroxocobalamin and carboxy-PTIO on non-adrenergic non-cholinergic nerve mediated relaxations of the rat duodenum.

Relaxation induced by NANC-nerve stimulation is reduced by nitric oxide synthase (NOS) inhibitors but not by superoxide generators or NO scavengers, casting doubts on the precise nature of the neurotransmitter being released by these nerves. The lack of effect of superoxide anion generators to inhibit nitrergic nerve-mediated relaxations has been attributed to the protective action of high tissue levels of superoxide dismutase (SOD). The effects of hydroquinone, hydroxocobalamin and carboxy-PTIO, three NO inactivators which do not depend on superoxide anion generation, upon nitrergic nerve-mediated relaxations of the rat proximal duodenum were determined in order to elucidate whether they are mediated by free NO. GABA and nicotine caused relaxations of isolated segments of the rat proximal duodenum in a concentration-dependent manner that were abolished by tetrodotoxin (TTX). Similarly, transmural electrical stimulation (TES) caused frequency-dependent relaxations that were also abolished by TTX. The NOS inhibitors L-NAME and L-NOARG reduced in a concentration-dependent manner nerve-mediated relaxations elicited by TES, nicotine and GABA. The effect of NOS inhibitors was prevented by L-arginine but not D-arginine. NO caused concentration-dependent relaxations that were not affected by TTX or L-NOARG but were abolished by hydroquinone, hydroxocobalamin and carboxy-PTIO. In contrast, these compounds failed to affect TES-, nicotine- and GABA-induced relaxations. The lack of effect of hydroquinone, hydroxocobalamin and carboxy-PTIO upon nerve-mediated relaxations was unaltered by pretreatment with the SOD irreversible inhibitor DETCA. The present findings show that nitrergic nerve-mediated relaxations of the rat duodenum are unaffected by NO inactivators that do not generate superoxide anion. It is suggested that either a NO-containing molecule that is unreactive with the inactivators tested is the inhibitory neurotransmitter released by nitrergic nerves or that NOS activity fulfills another role in nitrergic nerves which could be related to the release of an still unidentified transmitter.

Iron overloading inhibits dentine mineralization.

The present study reveals the inhibitory effect of iron intoxication on the process of dentine mineralization. Wistar rats were injected intraperitoneally with iron dextran at 0.88 g/kg body weight per day for 10 days during the period of odontogenesis. An age-matched group was injected intraperitoneally with bisodium etidronate (EHBP) at 20 mg/kg body weight per day for 10 days. Another age-matched group was treated with similar amounts of saline intraperitoneally and considered as control. At the end of the experimental period the animals intoxicated with iron exhibited non-mineralized dentine and mineralized bone. The animals treated with EHBP showed non-mineralized dentine and bone. These findings would suggest the existence of different mineralization mechanisms for bone and dentine.

The evolution of purinergic receptors involved in recognition of a blood meal by hematophagous insects

Many blood feeders use adenine nucleotides as cues for locating blood meal. Structure-activity relationship of adenine nucleotides as phagostimulants varies between closely-related species of blood feeders. It is suggested that a preexisting diverse pool of nucleotide-binding proteins present in all living cells, serves as a source of receptor proteins for the gustatory receptors involved in blood detection. It is proposed that the selection of any such nucleotide-binding protein is random.