Furosemide stress test and interstitial fibrosis in kidney biopsies in chronic kidney disease.

BACKGROUND: Interstitial fibrosis (IF) on kidney biopsy is one of the most potent risk factors for kidney disease progression. The furosemide stress test (FST) is a validated tool that predicts the severity of acute kidney injury (especially at 2 h) in critically ill patients. Since furosemide is secreted through the kidney tubules, the response to FST represents the tubular secretory capacity. To our knowledge there is no data on the correlation between functional tubular capacity assessed by the FST with IF on kidney biopsies from patients with chronic kidney disease (CKD). The aim of this study was to determine the association between urine output (UO), Furosemide Excreted Mass (FEM) and IF on kidney biopsies after a FST. METHODS: This study included 84 patients who underwent kidney biopsy for clinical indications and a FST. The percentage of fibrosis was determined by morphometry technique and reviewed by a nephropathologist. All patients underwent a FST prior to the biopsy. Urine volume and urinary sodium were measured in addition to urine concentrations of furosemide at different times (2, 4 and 6 h). We used an established equation to determine the FEM. Values were expressed as mean, standard deviation or percentage and Pearson Correlation. RESULTS: The mean age of the participants was 38 years and 44% were male. The prevalence of diabetes mellitus, hypertension and diuretic use was significantly higher with more advanced degree of fibrosis. Nephrotic syndrome and acute kidney graft dysfunction were the most frequent indications for biopsy. eGFR was inversely related to the degree of fibrosis. Subjects with the highest degree of fibrosis (grade 3) showed a significant lower UO at first hour of the FST when compared to lower degrees of fibrosis (p = 0.015). Likewise, the total UO and the FEM was progressively lower with higher degrees of fibrosis. An inversely linear correlation between FEM and the degree of fibrosis (r = - 0.245, p = 0.02) was observed. CONCLUSIONS: Our findings indicate that interstitial fibrosis correlates with total urine output and FEM. Further studies are needed to determine if UO and FST could be a non-invasive tool to evaluate interstitial fibrosis. TRIAL REGISTRATION: ClinicalTrials.gov NCT02417883.

CDP-choline circumvents mercury-induced mitochondrial damage and renal dysfunction.

Heavy metal ions are known to produce harmful alterations on kidney function. Specifically, the accumulation of Hg2+ in kidney tissue may induce renal failure. In this work, the protective effect of CDP-choline against the deleterious effects induced by Hg2+ on renal function was studied. CDP-choline administered ip at a dose of 125 mg/kg body weight prevented the damage induced by Hg2+ administration at a dose of 3 mg/kg body weight. The findings indicate that CDP-choline guards mitochondria against Hg2+ -toxicity by preserving their ability to retain matrix content, such as accumulated Ca2+ . This nucleotide also protected mitochondria from Hg2+ -induced loss of the transmembrane electric gradient and from the generation of hydrogen peroxide and membrane TBARS. In addition, CDP-choline avoided the oxidative damage of mtDNA and inhibited the release of the interleukins IL-1 and IL6, recognized as markers of acute inflammatory reaction. After the administration of Hg2+ and CDP, CDP-choline maintained nearly normal levels of renal function and creatinine clearance, as well as blood urea nitrogen (BUN) and serum creatinine.

Rehydration with soft drink-like beverages exacerbates dehydration and worsens dehydration-associated renal injury.

Recurrent dehydration, such as commonly occurs with manual labor in tropical environments, has been recently shown to result in chronic kidney injury, likely through the effects of hyperosmolarity to activate both vasopressin and aldose reductase-fructokinase pathways. The observation that the latter pathway can be directly engaged by simple sugars (glucose and fructose) leads to the hypothesis that soft drinks (which contain these sugars) might worsen rather than benefit dehydration associated kidney disease. Recurrent dehydration was induced in rats by exposure to heat (36°C) for 1 h/24 h followed by access for 2 h to plain water (W), a 11% fructose-glucose solution (FG, same composition as typical soft drinks), or water sweetened with noncaloric stevia (ST). After 4 wk plasma and urine samples were collected, and kidneys were examined for oxidative stress, inflammation, and injury. Recurrent heat-induced dehydration with ad libitum water repletion resulted in plasma and urinary hyperosmolarity with stimulation of the vasopressin (copeptin) levels and resulted in mild tubular injury and renal oxidative stress. Rehydration with 11% FG solution, despite larger total fluid intake, resulted in greater dehydration (higher osmolarity and copeptin levels) and worse renal injury, with activation of aldose reductase and fructokinase, whereas rehydration with stevia water had opposite effects. In animals that are dehydrated, rehydration acutely with soft drinks worsens dehydration and exacerbates dehydration associated renal damage. These studies emphasize the danger of drinking soft drink-like beverages as an attempt to rehydrate following dehydration.

Synergistic effect of uricase blockade plus physiological amounts of fructose-glucose on glomerular hypertension and oxidative stress in rats.

Fructose in sweetened beverages (SB) increases the risk for metabolic and cardiorenal disorders, and these effects are in part mediated by a secondary increment in uric acid (UA). Rodents have an active uricase, thus requiring large doses of fructose to increase plasma UA and to induce metabolic syndrome and renal hemodynamic changes. We therefore hypothesized that the effects of fructose in rats might be enhanced in the setting of uricase inhibition. Four groups of male Sprague-Dawley rats (n = 7/group) were studied during 8 wk: water + vehicle (V), water + oxonic acid (OA; 750 mg/k BW), sweetened beverage (SB; 11% fructose-glucose combination) + V, and SB + OA. Systemic blood pressure, plasma UA, triglycerides (TG), glucose and insulin, glomerular hemodynamics, renal structural damage, renal cortex and liver UA, TG, markers of oxidative stress, mitDNA, fructokinase, and fatty liver synthase protein expressions were evaluated at the end of the experiment. Chronic hyperuricemia and SB induced features of the metabolic syndrome, including hypertension, hyperuricemia, hyperglycemia, and systemic and hepatic TG accumulation. OA alone also induced glomerular hypertension, and SB alone induced insulin resistance. SB + OA induced a combined phenotype including metabolic and renal alterations induced by SB or OA alone and in addition also acted synergistically on systemic and glomerular pressure, plasma glucose, hepatic TG, and oxidative stress. These findings explain why high concentrations of fructose are required to induce greater metabolic changes and renal disease in rats whereas humans, who lack uricase, appear to be much more sensitive to the effects of fructose.

Detection of IS6110 and HupB gene sequences of Mycobacterium tuberculosis and bovis in the aortic tissue of patients with Takayasu's arteritis.

BACKGROUND: Takayasu's arteritis (TA) is a chronic inflammatory disease affecting the large arteries and their branches; its etiology is still unknown. In individuals suffering from TA, arterial inflammation progresses to stenosis and/or occlusion, leading to organ damage and affecting survival. Relation of TA with Mycobacterium tuberculosis has been known, but there have been only a few systematic studies focusing on this association. The IS6110 sequence identifies the Mycobacterium tuberculosis complex and the HupB establishes the differences between M. tuberculosis and M. bovis. Our objective was to search the presence of IS6110 and HupB genes in aorta of patients with TA. METHODS: We analyzed aorta tissues embedded in paraffin from 5760 autopsies obtained from our institution, we divided the selected samples as cases and controls; CASES: aortic tissues of individuals with Takayasu's arteritis. Control positive: aortic tissues (with tuberculosis disease confirmed) and control negative with other disease aortic (atherosclerosis). RESULTS: Of 181 selected aorta tissues, 119 fulfilled the corresponding criteria for TA, TB or atherosclerosis. Thus 33 corresponded to TA, 33 to tuberculosis (TB) and 53 to atherosclerosis. The mean age was 22 ± 13, 41 ± 19, and 57 ± 10, respectively. IS6110 and HupB sequences were detected in 70% of TA tissues, 82% in tuberculosis, and in 32% with atherosclerosis. Important statistical differences between groups with TA, tuberculosis versus atherosclerosis (p = 0.004 and 0.0001, respectively) were found. CONCLUSION: We identified a higher frequency of IS6110 and HupB genes in aortic tissues of TA patients. This data suggests that arterial damage could occur due to previous infection with M. tuberculosis.

Uric acid-induced endothelial dysfunction is associated with mitochondrial alterations and decreased intracellular ATP concentrations.

BACKGROUND/AIMS: Endothelial dysfunction is associated with mitochondrial alterations. We hypothesized that uric acid (UA), which can induce endothelial dysfunction in vitro and in vivo, might also alter mitochondrial function. METHODS: Human aortic endothelial cells were exposed to soluble UA and measurements of oxidative stress, nitric oxide, mitochondrial density, ATP production, aconitase-2 and enoyl Co-A hydratase-1 expressions, and aconitase-2 activity in isolated mitochondria were determined. The effect of hyperuricemia induced by uricase inhibition in rats on renal mitochondrial integrity was also assessed. RESULTS: UA-induced endothelial dysfunction was associated with reduced mitochondrial mass and ATP production. UA also decreased aconitase-2 activity and lowered enoyl CoA hydratase-1 expression. Hyperuricemic rats showed increased mitDNA damage in association with higher levels of intrarenal UA and oxidative stress. CONCLUSIONS: UA-induced endothelial dysfunction is associated with mitochondrial alterations and decreased intracellular ATP. These studies provide additional evidence for a deleterious effect of UA on vascular function that could be important in the pathogenesis of hypertension and vascular disease.

Sildenafil treatment prevents glomerular hypertension and hyperfiltration in rats with renal ablation.

BACKGROUND: Sildenafil treatment ameliorates progressive renal injury resulting from extensive renal ablation; however, modifications induced by sildenafil in the glomerular hemodynamic pathophysiology of the remnant kidney have not been investigated. AIM: To determine the effects of sildenafil in the glomerular microcirculation and their relation to histological damage in the renal ablation model. METHODS: Micropuncture studies were performed 60 days after 5/6 nephrectomy in rats that received no treatment, sildenafil (5 mg/kg/day) and reserpine, hydralazine and hydrochlorothiazide to maintain the blood pressure within normal levels. Sham-operated rats untreated and treated with sildenafil served as controls. RESULTS: As expected, renal ablation induced systemic and glomerular hypertension, hyperfiltration, proteinuria, glomerulosclerosis and tubulointerstitial inflammatory damage in the remnant kidney. Sildenafil treatment prevented single-nephron hyperfiltration and hypertension, suppressed renal arteriolar remodeling, ameliorated systemic hypertension and proteinuria, increased urinary excretion of cGMP and NO(2)(-)/NO(3)(-), decreased oxidative stress and improved histological damage in the remnant kidney. Normalization blood pressure with reserpine, hydralazine and hydrochlorothiazide did not modify glomerular hemodynamics, proteinuria or histological changes induced by renal ablation. CONCLUSIONS: Beneficial effects of sildenafil in the remnant kidney are associated with a reduction in the arteriolar remodeling, renal inflammatory changes and prevention of changes in the glomerular microcirculation.

Contribution of renal purinergic receptors to renal vasoconstriction in angiotensin II-induced hypertensive rats.

To investigate the participation of purinergic P2 receptors in the regulation of renal function in ANG II-dependent hypertension, renal and glomerular hemodynamics were evaluated in chronic ANG II-infused (14 days) and Sham rats during acute blockade of P2 receptors with PPADS. In addition, P2X1 and P2Y1 protein and mRNA expression were compared in ANG II-infused and Sham rats. Chronic ANG II-infused rats exhibited increased afferent and efferent arteriolar resistances and reductions in glomerular blood flow, glomerular filtration rate (GFR), single-nephron GFR (SNGFR), and glomerular ultrafiltration coefficient. PPADS restored afferent and efferent resistances as well as glomerular blood flow and SNGFR, but did not ameliorate the elevated arterial blood pressure. In Sham rats, PPADS increased afferent and efferent arteriolar resistances and reduced GFR and SNGFR. Since purinergic blockade may influence nitric oxide (NO) release, we evaluated the role of NO in the response to PPADS. Acute blockade with N(ω)-nitro-l-arginine methyl ester (l-NAME) reversed the vasodilatory effects of PPADS and reduced urinary nitrate excretion (NO(2)(-)/NO(3)(-)) in ANG II-infused rats, indicating a NO-mediated vasodilation during PPADS treatment. In Sham rats, PPADS induced renal vasoconstriction which was not modified by l-NAME, suggesting blockade of a P2X receptor subtype linked to the NO pathway; the response was similar to that obtained with l-NAME alone. P2X1 receptor expression in the renal cortex was increased by chronic ANG II infusion, but there were no changes in P2Y1 receptor abundance. These findings indicate that there is an enhanced P2 receptor-mediated vasoconstriction of afferent and efferent arterioles in chronic ANG II-infused rats, which contributes to the increased renal vascular resistance observed in ANG II-dependent hypertension.

Relationship between angiotensin II receptor expression and cardiovascular risk factors in Mexican patients with coronary occlusive disease.

The density of Angiotensin II (Ang) receptors on tissue surfaces is regulated by multiple hormones, cytokines and metabolic factors and is profoundly affected by various pathological conditions, such as age, diet and environmental conditions. The participation of several cardiovascular risk factors in the regulation of Angiotensin II receptor expression has been incompletely studied. We performed an ex-vivo study with human aortic postsurgical specimens to test the hypothesis that Ang AT1 and AT2 receptor expression in human aortic arteries is associated with the presence of cardiovascular risk factors. We included 31 Mexican patients with coronary artery disease. We evaluated Angiotensin II receptor expression by immunostaining and angiotensin converting enzyme insertion/deletion (ACE I/D) polymorphisms by polymerase chain reaction. AT1 and AT2 receptor expression was increased in the aortic segments from the cardiovascular patients compared with control arteries and in patients with the DD genotype. There was a correlation between increased AT1 receptor expression and the number of cardiovascular risk factors present in the patient. Furthermore, reduction of AT1 expression correlated with the number of drug combinations used in the patients. These correlations were not present with respect to AT2 receptor expression. We suggest that increased AT1 receptor expression is associated with the DD genotype. Thus the presence of several cardiovascular risk factors as well as DD genotype, induce AT1 expression increasing the probability to develop coronary occlusive disease.

Interleukin 8 Is Overexpressed in Acute Rejection in Kidney Transplant Patients.

The main complication associated with renal graft loss is immune rejection. The gold standard for the diagnosis of renal graft rejection is percutaneous renal biopsy, which is expensive and can lead to complications. Inflammation is one of the main pathogenic pathways in allograft rejection, and urine samples seem to be efficient windows to explore the allograft condition with a high cost-benefit ratio. This study aimed to evaluate the messenger ribonucleic acid (mRNA) profile expression pattern for interleukin (IL) 2, IL-4, IL-6, IL-8, and IL-10; tumor necrosis factor alfa; gamma interferon; and transforming growth factor ß1 in the urine renal cells of patients with a diagnosis of humoral rejection and patients with a diagnosis of normal biopsy. METHODS: An observational, cross-sectional analytical study was performed. All kidney transplants were performed at the Organ Transplant Department between 2018 and 2019. Also, a healthy control with a normal blood test and no apparent infection was included. mRNA from urine samples and biopsies was isolated, and the expression of interleukins was analyzed in PCR real time. Data were analyzed by Shapiro-Wilk and Kruskal-Wallis tests. RESULTS: The proinflammatory IL expression pattern in urine samples of kidney rejection group showed overexpression for IL-8 (P = .0001). No differences were observed in the rest of the interleukins analyzed. When we compared the results in the rejected versus not rejected transplanted patients with a group of apparently healthy subjects, the difference remains consistent. Thus, mRNA of IL-8 could function as a diagnostic tool in cases of chronic damage secondary to fibrosis.

Analyses of Programmed Cell Death Protein 1 in High Immunologic-Risk Transplant Patients.

Kidney transplant (KT) is the first therapeutic option for most patients with chronic renal failure that requires renal function replacement. The main complication associated with renal graft loss is immune rejection. The T regulatory pathways play a key role in this process, and abnormalities in some of these molecules could participate in the graft rejection. In this paper, our group performed an exploratory analysis of the behavior of the coinducible molecules (CD28, CTLA-4, ICOS, PD-1) in patients with KT rejection and control KT patients without rejection. The Mann-Whitney U test, used for 2 groups, showed significant differences (P = .0005), indicating that PD-1 is underexpressed in patients with allograft rejection. No differences were found in CD28+, regulatory T cells (T reg), CTLA-4, and ICOS, so we are proposing that PD-1 is a key player in the immunotolerance phenomenon and its underexpression participates in the rejection process. More research needs to be performed on this topic.

Restricted Water Intake and Hydration with Fructose-Containing Beverages during Infancy Predispose to Aggravate an Acute Renal Ischemic Insult in Adolescent Rats.

We aimed to investigate the effects of chronic fluid restriction and hydration with a sweetened beverage (SB) in rats from weaning until adolescence, in a posterior acute kidney injury (AKI) event induced by ischemia-reperfusion (I/R). We followed 5 groups of weaning rats: control group (C); two groups with 22 h/day fluid restriction, a group hydrated for two hours with water (-W) and a group hydrated with SB; one group receiving SB ad libitum all day (+SB); and one group in which water consumption was increased using a gel diet. The rats that reached adolescence were submitted to I/R. Fluid restriction and/or SB hydration induced mild renal alterations that were significantly accentuated in the -SB group and resulted in worse outcomes after I/R-induced AKI that resulted in a catastrophic fall in creatinine clearance and diffuse acute tubular necrosis. In summary, low tap water intakes, as well as SB intake in infancy, prompt kidney worse outcomes in a later event of AKI during adolescence and both insults magnify kidney damage. Studies on hydration habits in children are recommended to disclose the potentially harmful effects that those behavioral patterns might carry to future renal health.

Chronic Kidney Disease in Children Aged 6-15 Years and Associated Risk Factors in Apizaco, Tlaxcala, Mexico, a Pilot Study.

INTRODUCTION: Tlaxcala, a small state in central Mexico, has the highest prevalence of chronic kidney disease (CKD) deaths in population aged 5-14 in Mexico, most of them with unknown etiology. OBJECTIVE: To determine the prevalence of CKD in apparently healthy pediatric population in Apizaco, Tlaxcala. METHODS: A cross-sectional pilot study was carried out in children deemed as healthy; subjects with previous diagnosis of CKD were excluded. Informed consent was obtained in all cases. A physical examination was performed, a questionnaire was applied. Blood and urine samples were obtained for serum creatinine, urinalysis, and microalbumin/creatinine ratio. A second and third evaluation was performed after 6 and 18 months in those found with urinary anomalies/CKD to confirm the diagnosis. RESULTS: One hundred and nine subjects completed physical examination, which are the biological samples. Median age was 12 years. CKD stage 2 was confirmed in 5 subjects in the sixth month confirmation visit (4.6%). One patient accepted renal biopsy and Alport Syndrome was found. In a robust multivariate analysis, the risk factors related to reduction in the glomerular filtration rate were males -5.15 mL/min/1.73 m2 (p = 0.002), older participants as by -1.58 mL/min/1.73 m2 per year (p < 0.0001), and among participants living close to a river -3.76 mL/min/1.73 m2 (p = 0.033). DISCUSSION/CONCLUSION: The prevalence of CKD in the population studied in Apizaco Tlaxcala was confirmed in 4.6 cases per 100 inhabitants between 6 and 15 years. Males, older age, and living close to a river were the risk predictive factors. More studies are needed to determine the causes of the high CKD prevalence in this population.

Treatment with the xanthine oxidase inhibitor febuxostat lowers uric acid and alleviates systemic and glomerular hypertension in experimental hyperuricaemia.

BACKGROUND: Experimentally-induced hyperuricaemia [due to inhibition of uricase with oxonic acid (OA)] in rats causes hypertension and renal alterations which can be prevented by lowering uric acid (UA) with allopurinol. Febuxostat (Fx), an investigational, nonpurine and selective xanthine oxidase inhibitor, is a more effective UA-lowering agent than allopurinol. We therefore tested the hypothesis that Fx might be useful in treating hyperuricemia-induced hypertension and renal damage. METHODS: Four groups of male rats were studied: OA (750 mg/kg by daily gavage) was given for 8 weeks and Fx (5-6 mg/kg/day in drinking water; OA+Fx: n = 10) or placebo (OA+P: n = 11) were administered for 4 weeks beginning at 4 weeks after initiation of the study. Two groups of normal (N) rats were studied as controls (N+P and N+Fx: n = 10/group). Systolic blood pressure (SBP) and fasting plasma UA were measured in all animals at baseline and at 4 and 8 weeks. Glomerular haemodynamics by micropuncture techniques were determined at 8 weeks followed by histological evaluation of glomerular and afferent arteriole morphologies. RESULTS: In OA-induced hyperuricaemic rats, Fx lowered UA and ameliorated systemic and glomerular hypertension as well as mesangial matrix expansion and the development of preglomerular arteriolar disease as indicated by a reduction of the arteriolar area and media-to-lumen ratio. In normal rats, Fx tended to lower UA and had no effect on blood pressure, renal hemodynamics and afferent arteriole morphology. CONCLUSION: These results suggest that Fx merits further evaluation for the treatment of hypertension and renal alterations induced by hyperuricaemia.

Effect of febuxostat on the progression of renal disease in 5/6 nephrectomy rats with and without hyperuricemia.

BACKGROUND/AIMS: The effect of febuxostat (Fx), a non-purine and selective xanthine oxidase inhibitor, on glomerular microcirculatory changes in 5/6 nephrectomy (5/6 Nx) Wistar rats with and without oxonic acid (OA)-induced hyperuricemia was evaluated. METHODS: Four groups were studied: 5/6 Nx+vehicle (V)+placebo (P) (n = 7); 5/6 Nx+V+Fx (n = 8); 5/6 Nx+OA+P (n = 6) and 5/6 Nx+OA+Fx (n = 10). OA (750 mg/kg/day, oral gavage) and Fx (3-4 mg/kg/day, drinking water) were administered for 4 weeks. Systolic blood pressure, proteinuria and plasma uric acid were measured at baseline and at the end of 4 weeks. Measurement of glomerular hemodynamics and evaluation of histology were performed at the end of 4 weeks. RESULTS: 5/6 Nx+OA+P rats developed hyperuricemia, renal vasoconstriction and glomerular hypertension in association with further aggravation of afferent arteriolopathy compared to 5/6 Nx+V+P. Fx prevented hyperuricemia in 5/6 Nx+OA+Fx rats and ameliorated proteinuria, preserved renal function and prevented glomerular hypertension in both 5/6 Nx+V+Fx and 5/6 Nx+OA+Fx groups. Functional improvement was accompanied by preservation of afferent arteriolar morphology and reduced tubulointerstitial fibrosis. CONCLUSION: Fx prevented renal injury in 5/6 Nx rats with and without coexisting hyperuricemia. Because Fx helped to preserve preglomerular vessel morphology, normal glomerular pressure was maintained even in the presence of systemic hypertension.

Probiotic supplements prevented oxonic acid-induced hyperuricemia and renal damage.

Hyperuricemia is highly prevalent and especially common in subjects with metabolic, cardiovascular and renal diseases. In chronic kidney disease, hyperuricemia is extremely common, and uric acid (UA) excretion relies on gut uricolysis by gut microbiota. Current therapy for lowering serum UA includes drugs that may produce undesired secondary effects. Therefore, this pilot study was designed to evaluate the potential of two probiotic supplements to reduce systemic uric acid concentrations. Secondary objectives were to assess whether the hypouricemic effect related to a therapeutic benefit on the hyperuricemia-induced renal damage and hypertension. Analysis of fecal microbiota was also performed. Groups of 6 rats each were followed for 5 weeks and allocated in the following treatment groups: C = Control; HU-ND = Oxonic acid-induced hyperuricemia (HU) +regular diet; HU-P = HU+placebo; HU-F1 = HU+ probiotics formula 1 and HU-F2 = HU+ probiotics formula 2. We confirmed that oxonic acid-induced hyperuricemia produced hypertension and renal functional and structural changes, along with modest changes in the overall composition of fecal microbiota. Both probiotic-containing diets prevented HU, elevated UA urinary excretion and intrarenal UA accumulation induced by oxonic acid. The hypouricemic effect conferred by probiotic supplementation also prevented the renal changes and hypertension caused by hyperuricemia. However, probiotic treatment did not restore the fecal microbiota. In conclusion, we demonstrated for the first time the ability of probiotics containing uricolytic bacteria to lower serum uric acid in hyperuricemic animals with beneficial consequences on blood pressure and renal disease. As probiotics supplements are innocuous for human health, we recommend clinical studies to test if probiotic supplements could benefit hyperuricemic individuals.

Mitochondrial permeability transition relevance for apoptotic triggering in the post-ischemic heart.

Dysfunction of mitochondrial calcium homeostasis transforms this cation from a key regulator of mitochondrial function, into a death effector during post-ischemic reperfusion. High intramitochondrial calcium and prevailing cellular conditions favor the opening of the mitochondrial permeability transition pore (mPTP), that induces mitochondrial swelling and provides a mechanism for cytochrome c release, a hallmark signal protein of the mitochondrial apoptosis pathway; indeed, a second mechanism induced by pro-apoptotic BAX protein, could account for cytochrome c leak in the post-ischemic heart. The present study was undertaken to determine which one of these mechanisms triggers the mitochondrial apoptosis pathway in the reperfused heart. To accomplish this goal we prevented the opening of the mPTP in such hearts, by diminishing calcium overload with Ru360, a specific mitochondrial calcium uniporter inhibitor. We found that mPTP opening in reperfused hearts increased along with reperfusion time and concurs with cytochrome c release from mitochondria. Maximal cytochrome c release correlated with mitochondrial dysfunction and complete NAD+ deletion. Fully inserted BAX was detected early after reperfusion and remained unchanged during the evaluated reperfusion times. Remarkably, heart perfusion with Ru360, inhibited mPTP opening and BAX docking into the mitochondrial membranes, suggesting a mPTP upstream role on BAX migration/insertion.

Lead exposure effect on angiotensin II renal vasoconstriction.

Low levels of chronic lead exposure can produce hypertension and endothelial dysfunction, which could be associated with oxidative stress, changes in vascular tone and an imbalance of endothelial-derived vasoconstriction and vasodilator factors. The aim was to investigate the effect of chronic lead-exposure on angiotensin II-induced vasoconstriction in isolated perfused kidney and microvessels. Male Wistar rats (230-250 g) were treated for 12 weeks with lead acetate (100 ppm, Pbgroup) or pure water (control group). We evaluated the vascular reactivity in the kidneys and renal microvessels in the presence and absence of N(omega)-nitro-L-arginine methyl ester (L-NAME) in both groups. The nitrite concentration in renal perfusate was measured as an index of NO released, renal abundance of 3-nitrotyrosine was measured as well as endothelial NO synthase (eNOS) expression. Oxidative stress was measured by using the oxidative fluorescence dye dihydroethidium (DHE) to evaluate in situ production of superoxide and identified by confocal microscopy. Lead-exposure significantly increased blood pressure, eNOS protein expression, oxidative stress and vascular reactivity to angiotensin II. L-NAME potentiated vascular response to angiotensin II in control group but had no effect on the Pb-group. Nitrites released from the kidney of lead-group was lower compared to the control group while 3-nitrotyrosine was higher. This data suggest that lead-induced hypertension could be caused partially by an altered NOsystem.

Vasopressin Mediates the Renal Damage Induced by Limited Fructose Rehydration in Recurrently Dehydrated Rats.

Recurrent dehydration and heat stress cause chronic kidney damage in experimental animals. The injury is exacerbated by rehydration with fructose-containing beverages. Fructose may amplify dehydration-induced injury by directly stimulating vasopressin release and also by acting as a substrate for the aldose reductase-fructokinase pathway, as both of these systems are active during dehydration. The role of vasopressin in heat stress associated injury has not to date been explored. Here we show that the amplification of renal damage mediated by fructose in thermal dehydration is mediated by vasopressin. Fructose rehydration markedly enhanced vasopressin (copeptin) levels and activation of the aldose reductase-fructokinase pathway in the kidney. Moreover, the amplification of the renal functional changes (decreased creatinine clearance and tubular injury with systemic inflammation, renal oxidative stress, and mitochondrial dysfunction) were prevented by the blockade of V1a and V2 vasopressin receptors with conivaptan. On the other hand, there are also other operative mechanisms when water is used as rehydration fluid that produce milder renal damage that is not fully corrected by vasopressin blockade. Therefore, we clearly showed evidence of the cross-talk between fructose, even at small doses, and vasopressin that interact to amplify the renal damage induced by dehydration. These data may be relevant for heat stress nephropathy as well as for other renal pathologies due to the current generalized consumption of fructose and deficient hydration habits.