Urinary TBARS Research Articles

Physical Training Is a Potential Modifier of Risk for Contrast-Induced Acute Kidney Injury in Diabetes Mellitus.

Background Iodinated contrast (IC) is a leading cause of hospital-based acute kidney injury (AKI). Contrast-induced acute kidney injury (CI-AKI) is a decline in renal function due to iodinated contrast administration and occurs more frequently in individuals with increasingly common risk factors, such as diabetes mellitus (DM). Physical training (PT) can have renoprotective effects on CI-AKI in diabetic nephropathy. The aim of this study was to evaluate the injury in kidneys of diabetic rats submitted to treatment with IC, evaluating the impact of PT on hemodynamics and renal function in addition to oxidative profile in diabetic rats submitted to IC-AKI. Materials and Methods Adult male Wistar rats are randomized into four groups: citrate (n = 7): control group, citrate buffer (streptozotocin-STZ vehicle), intravenous tail (iv), single dose; DM (n = 7): STZ, 60 mg/kg, iv, single dose; DM+IC (n = 7): DM rats treated with IC (sodium meglumine ioxithalamate, 6 mL/kg, intraperitoneal (ip), single dose); DM+IC+PT (n = 7): DM rats treated with IC as mentioned and submitted to physical training. Renal function parameters (inulin clearance, neutrophil gelatinase-associated lipocalin (NGAL), serum creatinine, and urinary albumin), hemodynamics (renal blood flow and renal vascular resistance), and oxidative profile (urinary peroxides, urinary TBARS, urinary nitric oxide, and renal tissue thiols) were evaluated. Results It was possible to observe a decrease in inulin clearance, renal blood flow, and thiols in renal tissue accompanied by an increase in urinary flow, serum creatinine, urinary albumin, renal vascular resistance, urinary peroxides, urinary nitrate, and TBARS in the DM group compared to the citrate group. The DM+IC group showed a reduction in inulin clearance, and the renal dysfunction was also seen by the increased NGAL. Renal hemodynamics and oxidative profile compared were also worsened in the DM group. PT improved renal function by increasing renal blood flow and thiol levels in renal tissue and reduced renal vascular resistance, metabolites of reactive oxygen, nitrogen species, and lipid peroxidation in the DM+IC+PT group compared to DM+IC. Conclusions Our results confirmed that DM induction increases renal vulnerability to the toxicity of IC and an association between DM with IC predisposes to severe AKI with reduced renal function alongside with renal hemodynamic alterations and oxidative mechanism of injury. The PT showed a renoprotective effect in DM animals subjected to damage with IC by modulating renal hemodynamics and oxidative profile, confirming a potential to modify the risk of CI-AKI when diabetes mellitus is present.

Attenuation of Oxidative Stress Contributes to Sex Differences in Renal Function between Adult Dahl S Rats

Hypertension is a major contributor to morbidity and mortality worldwide. In the United States, age‐matched prevalence of hypertension from 2015–2016 was 45.4%. Sex differences in hypertension are well established. Before 45 years of age, more men have hypertension than women, whereas after 65 years of age, more women have hypertension than men. However, many studies in hypertension show results only in male animals. This study uses male and female Dahl SS/jr rats to test the hypothesis that the ability to attenuate levels of oxidative stress in the kidney contributes to sex differences in renal injury and function. Male and female Dahl S rats were mated non‐consanguineously and offspring were weaned at four weeks of age. All animals in all studies were on a 0.3% NaCl diet, and measurements were made in offspring at three and six months of age. Systolic BP (n=6–10/group, tail cuff) at three and six months did not differ between sexes, although male animals did exhibit a significant increase over this period (3 months; male: 159±9 mmHg; female: 165±3 mmHg, p=0.6; 6 months; male: 180±4 mmHg; female: 173±12 mmHg, p=0.5). Urine was collected (n=7–20/group) for measurement of urinary protein (Bradford assay), nephrin, and kidney injury marker‐1 (KIM‐1). Females exhibited reduced renal injury at both three and six months of age as indicated by a significant reduction in both proteinuria (3 months, male: 102±8 mg/day; female: 55±5 mg/day, p<0.0001; 6 months, male: 238±25 mg/day; female: 44±7 mg/day, p=0.0001) and KIM‐1 (3 months, male: 24±2 ng/day; female: 18±2 ng/day, p=0.03; 6 months, male: 26±2 ng/day; female: 14±2 ng/day, p=0.001). Nephrin excretion was significantly lower in females at three months (Male: 107±13 ng/day; female: 59±6 ng/day, p=0.006), but this difference did not appear at six months of age (Male: 249±53 ng/day; female: 231±57 ng/day, p=0.8), suggesting that glomerular injury progressed in both sexes by this time. Targeted RNA sequencing was performed on RNA isolated from kidney using a custom renal injury/inflammation gene panel (n=32 genes), including Sod1, 2, and 3 genes. Females exhibited higher levels of the extracellular isoform Sod3 at three (male: 0.35±0.03 normalized CPM; female: 1.44±0.2, p=0.002) and six months (male: 0.41±0.05; female: 1.29±0.11, p=0.003). However, no significant differences were seen in expression of Sod1 or Sod2. Urinary TBARS was quantified as a measure of lipid peroxidation and was significantly lower in females at three months (Male: 65±3 μM/day; female: 44±4 μM/day, p=0.009), but a difference was not present at six months (Male: 91±16 μM/day; female: 77±12 μM/day, p=0.5). These data support the hypothesis that the ability to attenuate levels of extracellular oxidative stress in the kidney contributes to sex differences in renal injury and function.Support or Funding InformationThis research was supported by the National Institutes of Health under award numbers R01HL134711 (J.M. Sasser), F30DK118864 (H.R. Turbeville), and R01HL137673 (M.R. Garrett).

Diabetes Mellitus: a risk factor for drug toxicity.

To assess the effect of the antibiotic Gentamicin in an experimental model in the presence of Diabetes Mellitus through renal function and oxidative profile. Adult male Wistar rats were distributed into groups: Citrate; Gentamicin (Genta), (intraperitoneal, i.p. gentamicin, 100 mg/kg of body weight, once a day,5 days); DM (60 mg/kg of STZ (Streptozotocin), single dose, intravenously, i.v., diluted in citrate buffer); and DM+Genta. Physiological parameters, renal function (creatinine clearance), oxidative damage (peroxides and thiobarbituric acid reactive substances - urinary TBARS) and renal hemodynamics were evaluated. The Diabetes Mellitus group presented chronic hyperglycemia associated with loss of body weight, polyphagia, polydipsia and polyuria, in addition to reduced renal function and with an increase in oxidative metabolite excretion. Administration of gentamicin induced a reduction in renal blood flow and increased renal vascular resistance in healthy rats. The association of Diabetes Mellitus with gentamicin resulted in an additional reduction in renal function and elevation of oxidative metabolites, with increased renal vascular resistance. The existence of Diabetes Mellitus resulted in an elevation of gentamicin nephrotoxicity, thus confirming the risk factor for drug nephrotoxicity.

MnTBAP therapy attenuates the downregulation of sodium transporters in obstructive kidney disease.

Ureteral obstruction is associated with reduced expressions of renal sodium transporters, which contributes to impaired urinary concentrating capacity. In this study, we employed a synthetic mitochondrial superoxide dismutase 2 (SOD2) mimic MnTBAP to investigate the role of mitochondrial oxidative stress in modulating the sodium transporters in obstructive kidney disease. Following unilateral ureteral obstruction (UUO) for 7 days, a global reduction of sodium transporters including NHE3, NCC, NKCC2, and ENaCα was observed as determined by qRT-PCR, Western Blotting or immunohistochemistry. Among these sodium transporters, the downregulation of NHE3, NCC, and NKCC2 was partially reversed by MnTBAP treatment. In contrast, the reduction of ENaCα was not affected by MnTBAP. The β and γ subunits of ENaC were not significantly altered by ureteral obstruction or MnTBAP therapy. To further confirm the anti-oxidant effect of MnTBAP, we examined the levels of TBARs in the urine collected from the obstructed ureters of UUO mice and bladder of sham mice. As expected, the increment of urinary TBARs in UUO mice was entirely abolished by MnTBAP therapy, indicating an amelioration of oxidative stress. Meantime, we found that three types of SOD were all reduced in obstructed kidneys determined by qRT-PCR, which was unaffected by MnTBAP. Collectively, these results demonstrated an important role of mitochondrial oxidative stress in mediating the downregulation of sodium transporters in obstructive kidney disease.

Abstract P280: Butyric Acid Protects Against Aldosterone-salt-induced Hypertension and Renal Injury via Suppression of (Pro) Renin Receptor

Butyric acid (BA), a short-chain fatty acid derived from gut microbiota, exhibits beneficial effects on cardiovascular disease. We examined the potential antihypertensive and renal protective action of BA in a mouse model of aldosterone (Aldo)-salt-induced hypertension. C57/BL6 mice were subjected to Aldo-salt protocol consisting of minipump infusion of Aldo at 0.2 mg/kg/d plus 1% NaCl as drinking fluid, or in combination with minipump infusion of BA at 60 mg/kg/d. A 3-wk BA treatment lowered radio telemetry-measured MAP (146±3.8 vs. 133±2.9 mmHg, N=9, p<0.05), cardiac hypertrophy (6.7±0.32 vs. 5.4±0.28 mg/g body weight, N=9, p<0.05), 24-h urinary albumin (434±41vs. 218±22 μg/24h, N=9, p<0.01), the kidney hypertrophy (9.9±0.58 vs. 7.9±0.38 mg/g body weight, N=9, p<0.05), polyuria (19.5 ± 2.5 vs. 8.7±1.4 ml/24h, N=6, p<0.01), renal 8-isoprostane (0.74±0.14 vs. 0.35±0.05 pg/mg, N=6, p<0.05), and urinary TBARS (276±43 vs. 133±032 nmol/24h, N=9, p<0.05). The hematocrit was decreased by Aldo-salt, which was reversed by BA (Control: 50.0±0.4% vs. Aldo-salt: 46.2±0.8% vs. Aldo-salt + BA: 49.6±1.0%, all N=9, all p<0.05). Recent study has identified (pro) renin receptor as a key regulator of blood pressure. To examine the underlying mechanism of BA on Aldo-salt induced effect, we detected full length (pro) renin receptor (fPRR) and soluble PRR (sPRR) expression in vivo and in vitro in this study. The renal expression of fPRR and sPRR were increased 1.75-fold and 2.67-fold by Aldo-salt treatment, respectively, and these increases were both blocked by BA (1.75±0.08 vs. 0.93±0.14, N=6, p<0.01; 2.67±0.23 vs. 0.92±0.29, N=6, p<0.01, respectively). The Aldo-salt induced urinary sPRR excretion was also blunted by BA (Control: 1533±73 vs. Aldo-salt: 4800±365 vs. Aldo-salt + BA: 2615±138 pg/24h, all N=6, all p<0.05). In cultured mpkCCD cells, Aldo treatment induced protein expression of fPRR and sPRR, which were blocked by BA (fPRR: 1.25±0.09 vs. 0.73±0.08, N=5, p<0.01; sPRR: 2.33±0.35 vs. 1.03±0.15, N=5, p<0.01). Our results suggest that BA protected against Aldo-salt-induced hypertension and renal injury likely through suppression of PRR.

Inhibition of cholesterol and fatty acid synthesis by inhibiting SREBPs prevent diabetic nephropathy in db/db mice with type 2 diabetes (579.2)

Sterol Regulatory Element Binding Proteins (SREBPs) are master transcriptional regulators of fatty acid and cholesterol synthesis. In this study we examined the effects of SREBP inhibition on diabetic nephropathy. To this end we treated db/db mice of Betulin for 12 weeks.We found increased renal lipid accumulation in the kidneys of db/db mice mediated by increased expression of nuclear SREBP‐1 and SREBP‐2 protein. Treatment with Betulin prevented the upregulation of SREBPs in the kidney resulting in a correspondent reduction of kidney triglyceride and cholesterol accumulation. SREBP inhibition prevented the increases in urine albumin excretion (db/db: 59±13 µg/24hr; db/db‐betulin: 21±3 µg/24hr; p<0.05) and mesangial matrix expansion as shown by PAS staining of the kidneys of db/db mice (mesangial matrix index: db/db, 2.25±0.08; db/db‐betulin, 1.60±0.11; p<0.05). The treatment also prevented podocyte loss determined by staining with p57 and WT‐1. Furthermore, improvement in oxidative stress and inflammation was also observed in the treatment groups (urinary TBARS: db/db, 181±27 mmol/day vs. db/db‐betulin, 103±24 mmol/day, p<0.05; urinary MCP‐1: db/db, 146±22 mmol/day vs. db/db‐betulin, 83±15 mmol/day, p<0.05). To determine if Betulin has direct effects in kidney podocytes, we cultured human podocytes in high glucose conditions. Betulin prevented the high glucose induced increased the expression of SREBP‐1 and SREBP‐2 and lipid accumulation in human podocytes.Our study therefore indicates a renoprotective role for SREBP inhibition in diabetic nephropathy by preventing lipid accumulation and modulating oxidative stress and inflammation.Grant Funding Source: NIH, VA

Abstract 482: Renoprotective Effects of Exercise Training in Dahl Salt-sensitive Rats

Exercise training (Ex) slows the progression of hypertension and renal failure in some animal models. We and other groups have reported that Ex ameliorated hypertension and renal dysfunction in spontaneously hypertensive rats. Since little has been reported on the effects of Ex on renal disorder in salt-sensitive hypertension, we investigated the effects of Ex on blood pressure and renal function in Dahl salt-sensitive (DS) rats. Six-week-old male DS rats were divided into three groups: 1) Normal salt (0.5% NaCl) diet (NS) (NS group, n=10), 2) High salt (8% NaCl) diet (HS) (HS group, n=11), 3) HS plus moderate Ex with treadmill running (HE group, n=11). After 8 weeks, HS induced severe hypertension, and Ex did not affect systolic blood pressure (114±3, 209±6 and 205±8 mmHg in the NS, HS and HE groups, respectively). Plasma creatinine was significantly higher in the HS and HE groups than in the NS group, but not different between the HS and HE groups (0.22±0.01, 0.44±0.03 and 0.40±0.03 mg/dl). Urinary protein and albumin significantly increased in the HS and HE groups, but these were significantly lower in the HE group compared with the HS group (Protein: 15.9±1.6, 432.8±36.8 and 327.2±22.3 mg/day. Albumin: 3.9±0.8, 195.2±15.2 and 154.2±11.1mg/day). Urinary TBARS, an index of oxidative stress, significantly increased in the HS and HE groups, but this was significantly lower in the HE group compared with the HS group (0.30±0.02, 0.75±0.06 and 0.53±0.03 mg/day); there was no significant difference in plasma TBARS among all groups. CYP4A1 and CYP4A2 protein expressions in the outer medulla were significantly lowered by 37% and 48% in the HS group compared with the NS group, and were significantly elevated by 40% and 150% in the HE group compared with the HS group. There was no significant difference in cortical CYP4A1 protein expression among all groups. Although there was no difference in cortical CYP4A2 between the NS and HS groups, it was significantly increased by 35% in the HE group compared with the HS group. These results suggest that Ex attenuated HS-induced proteinuria independently of blood pressure or plasma creatinine level in DS rats. The improvement of oxidative stress and CYP4A expression in the kidneys may contribute to the renoprotective effects of Ex in DS rats.

The effect of dietary fat and omega‐3 fatty acids on whole body lipid oxidation

Lipid peroxidation of polyunsaturated fatty acids yields several electrophilic, reactive carbonyl metabolites. We hypothesized that an increased intake of omega‐3 fatty acids (n‐3) would lead to increased lipid peroxidation metabolites compared to a diet low in n‐3. As part of a randomized crossover dietary intervention trial, urinary markers of whole body lipid peroxidation were analyzed. Study subjects (n=15) included healthy, postmenopausal women who were fed a low‐fat diet (20% of energy from fat, LF) or low‐fat diet supplemented with 3% of energy from n‐3 fatty acids (LFn3) for 8 weeks. Urine was collected during two consecutive 24‐hour collections in the last two days of each diet. Whole body lipid peroxidation within each subject was measured via complementary methods. Analysis via urinary TBARS was followed by a sensitive HPLC separation and quantification of 2,4‐ dinitrophenylhydrazine‐reactive lipophilic aldehydes, including 4‐ hydroxynonenal, 4‐hydroxyhexenal, and other nonpolar aldehydes. The HPLC technique demonstrated that LFn3 resulted in increased levels of lipid peroxidation products by two‐fold relative to LF. These results show that increased n‐3 intake leads to increased production of in vivo secondary lipid peroxidation products. The impact of these results requires further investigation. This work was supported by the US DOD, NCI, NCRR, and Salmon of the Americas, Inc.Grant Funding Source: National Cancer Institute

Age-related changes in renal expression of oxidant and antioxidant enzymes and oxidative stress markers in male SHR and WKY rats

Oxidative stress has been hypothesized to play a role in aging and age-related disorders, such as hypertension. This study compared levels of oxidative stress and renal expression of oxidant and antioxidant enzymes in male normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) at different ages (3 and 12 months). In the renal cortex of 3-month old SHR increases in hydrogen peroxide (H 2O 2) were accompanied by augmented expression of NADPH oxidase subunit Nox4 and decreased expression of antioxidant enzymes SOD1 and SOD3. A further increase in renal H 2O 2 production and urinary TBARS was observed in 12-month old WKY and SHR as compared with 3-month old rats. Similarly, expressions of NADPH oxidase subunit p22 phox, SOD2 and SOD3 were markedly elevated with age in both strains. When compared with age-matched WKY, catalase expression was increased in 3-month old SHR, but unchanged in 12-month old SHR. Body weight increased with aging in both rat strains, but this increase was more pronounced in WKY. In conclusion, renal oxidative stress in 12-month old SHR is an exaggeration of the process already observed in the 3-month old SHR, whereas the occurrence of obesity in 12-month old normotensive rats may partially be responsible for the age-related increase in oxidative stress.

Two novel non-destructive biomarkers to assess PAH-induced oxidative stress and porphyrinogenic effects in crabs

Two novel, non-destructive assays were developed to evaluate contaminant-induced lipid peroxidation (thiobarbituric acid-reacting substances, TBARS, levels) and haem biosynthesis disruption (porphyrin excretion) in decapod crabs. A laboratory experiment was conducted whereby pie-crust crabs (Cancer novaezelandiae) were fed cockles (Austrovenus stutchburyi) collected from a contaminated and reference site and TBARS levels and porphyrin excretion determined using fluorometric analysis in urine samples. Pyrene metabolite levels were also measured in the same urine samples to assess polycyclic aromatic hydrocarbon (PAH) exposure. Contaminant-exposed crabs exhibited elevated urinary TBARS and porphyrin levels and a strong correlation was found between these two assays and the urinary pyrene metabolite concentrations. However, there was large within-treatment variability, which precluded a clear separation between the control and the impacted group. Nevertheless, consistency in the direction of the response shows that the biomarkers reflect pollutant levels and validates the use of these simple techniques from human medicine for environmental assessments.

Measurement of urinary excretion of 5-hydroxymethyluracil in human by GC/NICI/MS:: Correlation with cigarette smoking, urinary TBARS and etheno DNA adduct

5-Hydroxymethyluracil (5-HMU) is derived from radiation in addition to endogenous oxidative DNA damage and it is one of the most abundant DNA adducts. Human 5-HMU-DNA-glycosylase has been shown to repair this lesion. Whether urinary levels of 5-HMU is a valid biomarker for oxidative DNA damage in vivo has been investigated. However, controversial results on its relation to cigarette smoking were reported. To facilitate analysis of urinary 5-HMU in epidemiological studies, a highly sensitive and specific assay based on stable isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometry was developed. The limit of detection for N 1, N 3-bis(pentafluorobenzyl)-HMU is 10 fg (20 amol) ( S/ N = 4) injected on column and the limit of quantification in urine was 0.7 nM of 5-HMU. Using as little as 10 μL of human urine samples, levels of urinary 5-HMU in 21 healthy volunteers were accurately quantified. No correlation was observed between urinary 5-HMU levels and cigarette smoking. However, there was a statistically significant association between urinary levels of 5-HMU and thiobarbituric acid-reactive substances ( r = 0.71, p = 0.0003). In addition, urinary 5-HMU levels also correlated with urinary levels of 1, N 6-ethenoadenine ( r = 0.54, p = 0.01). These findings suggest that this assay should be valuable in assessing the role of urinary 5-HMU as a biomarker of oxidative DNA damage and repair.

BLOOD ANTIOXIDANT ENZYMES ADAPTATIONS AND OXIDATIVE STRESS BIOMARKERS STATUS DURING ENDURANCE AND INTERMITENT TRAINING.

393 Literature data have shown that training increases blood antioxidant capacity and reduces the exercise-induced oxidative stress. Although, most studies have been done using long term exercise and few data are available with intermitent training protocol and no one has shown the kinetics of these markers during the training period. Our purpose in this study was to examine the eritrocyte antioxidant adaptation induced between the two kinds of training and in addition we evaluated indexes of muscle damage measured by plasma creatine kinase (CK) and urinary Thiobarbituric Acid Reactive Substances (TBARs). Seventy male Wistar rats were divided in three groups, control sedentary group (CO), endurance trained group (ET), intermitent trained group (IT) and ran in a treadmill during eight weeks. Eritrocyte glutathione reductase(GR) and catalase(CAT) were assayed using spectophotometer methods. Whole blood CK and urinary TBARs were measured weekly using Reflotron (Boeringer Mannheim) and fluorimetrical method respectively. Our results showed that the ET antioxidant enzymes activity were higher at the end of the fourth week but at the end of the eighth week these activities were similar in both groups. On the other hand, CK and TBARs were closely related having their peaks between the fourth and fifth week in ET and IT group respectively, although these levels returned to the control levels in both groups at the end of the training period. Our data show that endurance and intermitent training increased antioxidant enzymes activities in eritrocytes, althought being faster in ET group and avoided free radical induced damage. Finantial support: FAPESP and CAPES