Neutrophil Gelatinase-associated Lipocalin Excretion Research Articles

Chronic circadian disruption impairs cardiovascular rhythms and leads to kidney disease in mice

Shift workers have an increased risk of developing hypertension and reduced kidney function. Disruption of behavioral or environmental circadian rhythms increases cardiometabolic disease risk in humans and animals. We hypothesized that chronic circadian disruption (CCD) impairs cardiovascular rhythms and increases renal injury markers. Male C57BL/6J mice starting at 8-week of age were maintained under a normal light/dark cycle (12-h light, 12-h dark, control) or a CCD protocol (10-h light, 10-h dark, T20) with ad libitum food and water for 10 weeks. T20 mice gained significantly more body weight compared to control mice. Control mice showed a diurnal variation in food intake, while T20 mice had similar food intake during both light and dark phases with more food consumed in the light phase compared to control mice. Mean arterial pressure (MAP), heart rate (HR), and locomotor activity were measured by telemetry after 10 weeks of CCD. MAP between the light and dark periods was significantly different in control mice (12:12 LD), while T20 mice (10:10 LD) lacked diurnal variation (control 119±3 vs.101±2 mm Hg, dark vs. light, respectively, p<0.001; T20 114±13 vs.113±12 mm Hg, dark vs. light, respectively, p=0.86; n=3-4). Control mice showed a diurnal variation in HR, whereas T20 mice did not show a light-dark difference (Control- 584±11 vs. 511±4 bpm, dark vs. light, respectively, p=0.001; T20- 537±14 vs. 526±5 bpm, dark vs. light, respectively, p=0.48; n=3-4). Dark phase HR was significantly decreased in T20 mice (p=0.01, control vs. T20). Control mice had a light-dark difference in activity, while T20 mice lacked diurnal variation in activity with significantly higher activity during the light phase (p=0.04, control vs. T20). Mice were placed in metabolic cages for urine collection during the light (control: ZT0-12; T20: ZT0-10) and dark (control: ZT12-0; T20: ZT10-0) phases. Urinary excretion of renal tubular injury markers albumin, neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM-1) were measured (ELISA). Control mice had significantly higher albumin and NGAL excretion in dark vs. light phases, while T20 mice lacked a significant diurnal pattern with significantly elevated albumin and NGAL during the light phase compared to control mice (albumin: control 1.00±0.23 vs. 0.09±0.05 ug/hr dark vs. light, p=0.04, control light vs. T20 light, p=0.04; NGAL: control 1410±223 vs. 89±58 pg/hr dark vs. light, p=0.02, control light vs.T20 light, p=0.04, n=4-6). CCD also blunted the diurnal rhythm of KIM-1 excretion (light cycle, p=0.04, n=3-6). The diurnal variation in sodium and potassium excretion was also lost in T20 mice. These results indicate that CCD impairs cardiovascular rhythms as well as diurnal excretion of electrolytes and renal injury markers, which may increase risk for kidney disease. P01HL136267, AHA 856877 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Analysis of Neutrophil Gelatinase-Associated Lipocalin in Type 2 Diabetes Mellitus Patients

Neutrophil Gelatinase-Associated Lipocalin (NGAL) is a small protein with a molecular weight of 21 kDa, belongs to the lipocalin protein family and functions as a growth factor and differentiation of various cell types such as renal tubular epithelial cells. This aim of study was to determine the level of NGAL in controlled and uncontrolled type 2 Diabetes Mellitus (DM) patients. This study was a cross sectional study involving 70 type 2 DM patients with 30 patients with controlled type 2 DM and 40 uncontrolled type 2 DM groups. HbA1c, urea, creatinine, and urinalysis data were measured. NGAL level was measured using the ELISA method. The statistical tests used were Chi-Square, unpaired T test, Mann-Whitney test and Spearman test. The test result is significant if the p-value <0.05. NGAL normally found in the circulation will be filtered by the glomerulus and reabsorbed by the epithelial cells of the proximal renal tubule. Increased levels of NGAL can be found 2-6 hours in the blood and urine after injury to the kidneys. Excretion of NGAL in blood occurs when there is damage to the epithelial cells of the proximal tubule of the kidney. There was a significant difference in NGAL levels in the controlled and uncontrolled groups of type 2 DM patients. Positive correlation indicates that the higher the HbA1c and creatinine levels, the higher the NGAL level. There was a significant difference in NGAL levels in the controlled and uncontrolled type 2 DM groups (25.09 ± 6.83 vs 112.54 ± 170.38 ng / mL, with p value <0.001). There was a positive correlation between NGAL and HbA1c levels with p value <0.001, r =0.507 and creatinine levels with p value <0.001, r =0.769. NGAL normally found in the circulation will be filtered by the glomerulus and reabsorbed by the epithelial cells of the proximal renal tubule. Increased levels of NGAL can be found 2-6 hours in the blood and urine after injury to the kidneys. Excretion of NGAL in blood occurs when there is damage to the epithelial cells of the proximal tubule of the kidney. There was a significant difference in NGAL levels in the controlled and uncontrolled groups of type 2 DM patients. Positive correlation indicates that the higher the HbA1c and creatinine levels, the higher the NGAL level.

MO406: Evolution of Kidney Damage Associated With Tobacco Consumption

Abstract BACKGROUND AND AIMS Tobacco consumption is associated with cancer, cardiovascular and respiratory diseases. Although new research relates tobacco use with the development of kidney damage, classic biomarkers, such as plasma creatinine, are not useful to detect this kidney injury [1]. In this sense, innovative diagnostic methods have been developed, such as early biomarkers capable of detecting subclinical kidney damage in the initial stages of the disease [2, 3]. In fact, studies carried out in our laboratory have shown that smoking patients suffer subclinical kidney damage evidenced by the excretion of these urinary biomarkers [4]. The aim of this work was to study the evolution of kidney damage associated with tobacco consumption using these biomarkers after monitoring smoking patients for 2 years. METHOD A longitudinal study was carried out that included 150 smokers and 150 non-smokers, without previous kidney damage and risk factors for kidney disease, from a Salamanca Health Centre (Spain). Urine samples were collected at the time of inclusion and after 2 years, and the biomarkers of subclinical kidney damage N-acetyl-β-D-glucosaminidase (NAG), neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1) and total proteins, were quantified using ELISA and colorimetric techniques. The data obtained were analyzed with the statistical software SPSS®. RESULTS The results showed a higher excretion of NAG, NGAL, KIM-1 and total proteins in smoker patients with respect to non-smoker ones, measured at baseline, which was maintained after 2 years. CONCLUSION Our research suggests that subclinical kidney damage associated with tobacco consumption would remain stable in the 2 years studied and could become chronic and it could constitute the basis for the development of chronic kidney failure.

MO390: Prognosis of Kidney Damage Associated With Tobacco Consumption in Former Smokers

Abstract BACKGROUND AND AIMS Tobacco is a risk factor for some cardiovascular and respiratory diseases, as well as for the development of some tumours. Furthermore, new research has shown evidence of the risk that smokers have of developing kidney disease [1]. On the other hand, biomarkers used in the clinical setting, such as plasma creatinine, are not useful to detect that injury. Considering this problem, the need to use biomarkers that provide a faster, more reliable and efficient diagnosis is clear. It is the case of biomarkers of subclinical kidney damage [2, 3]. In fact, studies from our laboratory have shown that smoking patients suffer subclinical kidney damage evidenced by the excretion of these novel urinary biomarkers [4]. The aim of this study was to evaluate the prognosis of kidney damage associated with tobacco in former smokers using these biomarkers. That is, to study the role that smoking cessation plays in the evolution of kidney injury. METHOD A clinical study was designed where three groups of patients, without previous kidney damage and risk factors for kidney disease, were included: i) non-smokers (control group), ii) smokers and iii) former smokers (according to the World Health Organization, a person who has not used tobacco in the last 6–12 months). They were recruited from a Salamanca Health Centre (Spain). Urine samples were collected and the biomarkers of subclinical kidney damage N-acetyl-β-D-glucosaminidase (NAG), neutrophil gelatinase-associated lipocalin (NGAL), kidney injure molecule 1 (KIM-1) and total proteins, were quantified by ELISA and colorimetric techniques. The data obtained were analysed with the statistical software SPSS®. RESULTS The results showed a higher excretion of NAG, NGAL, KIM-1 and total proteins in smokers with respect to non-smoker ones. Rather, this excretion was significantly lower in the groups of former smokers and non-smokers. CONCLUSION Our research suggests that subclinical kidney damage associated with tobacco consumption would improve its prognosis when patients quit smoking, being able to recover kidney function to similar levels to non-smokers. This finding supports that smoking cessation carries an important benefit for the health of smoking patients.

Value of urine/serum Neutrophil gelatinase-associated lipocalin ratio in distinguishing acute kidney injury from chronic kidney disease

ObjectiveThis study was performed to test the hypothesis that neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker would be helpful for differentiation acute kidney injury (AKI) from chronic kidney disease (CKD) in kidney malfunction patients from the nephrology department. MethodsThis retrospective study included 355 patients admitted from the nephrology department with modification of diet in renal disease estimated glomerular filtration rate (MDRD eGFR) < 60 ml/min/1.73 m2. The subjects were categorized into AKI group (n = 204) and CKD group (n = 151). A propensity-matched analysis, incorporating 17 variables, was performed to control potential selection bias. ResultsUrinary NGAL (uNGAL) level in the AKI group was higher than in the CKD group (372.10 (170.10–690.63) vs 88.10 (52.00–238.80), P < 0.001), but there was no significant difference in serum NGAL (sNGAL). Both sNGAL and uNGAL had a correlation with MDRD eGFR in total patients, AKI patients, and CKD patients. The propensity-matched analysis enrolled 75 patients in each group. In matched AKI group, sNGAL was lower (401.20 (239.10–616.00) vs 468.50 (305.00–709.40), P = 0.049) and uNGAL was elevated (284.00 (136.90–690.90) vs 203.70 (69.20–596.00), P = 0.032), compared with the matched CKD group. In all patients (n = 355), the ratio of uNGAL and sNGAL (u/s NGAL), fractional excretion of NGAL (Fe NGAL) discriminated AKI from CKD (area under the curve, 0.803 and 0.790, respectively). After stratified kidney function, the sub-analyses found that u/s NGAL and Fe NGAL were shown to differ substantially between the AKI group and CKD group (all P < 0.01). The u/s NGAL ratio always had the highest AUC area in the sub-analyses. Conclusionsu/s NGAL might be helpful to discriminate AKI from CKD in kidney malfunction patients admitted to the nephrology department. Further confirmatory studies might be warranted.

Effect of 0.9% NaCl compared to plasma-lyte on biomarkers of kidney injury, sodium excretion and tubular transport proteins in patients undergoing primary uncemented hip replacement \u2013 a randomized trial

BackgroundIsotonic saline (IS) is widely used to secure perioperative cardiovascular stability. However, the high amount of chloride in IS can induce hyperchloremic acidosis. Therefore, IS is suspected to increase the risk of acute kidney injury (AKI). Biomarkers may have potential as indicators.MethodsIn a double-blinded, placebo-controlled study, 38 patients undergoing primary uncemented hip replacement were randomized to IS or PlasmaLyte (PL). Infusion was given during surgery as 15 ml/kg the first hour and 5 ml/kg the following two hours. Urinary samples were collected upon admission and the day after surgery. As surgery was initiated, urine was collected over the course of 4 h. Hereafter, another urine collection proceeded until the morning. Urine was analyzed for markers of AKI neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1). Arterious and venous blood samples for measurements of pH and plasma electrolytes including chloride (p-Cl) were collected as surgery was initiated, at the end of surgery and the following morning.ResultsIS induced an increase in p-Cl (111 ± 2 mmol/L after IS and 108 ± 3 after PL, p = 0.004) and a decrease in pH (7.39 ± 0.02 after IS and 7.43 ± 0.03 after PL, p = 0.001). Urinary NGAL excretion increased in both groups (ΔNGAL: 5.5 [4.1; 11.7] μg/mmol creatinine p = 0.004 after IS vs. 5.5 [2.1;9.4] μg/mmol creatinine after PL, p < 0.001). No difference was found between the groups (p = 0.839). Similarly, urinary KIM-1 excretion increased in both groups (ΔKIM-1: IS 115.8 [74.1; 156.2] ng/mmol creatinine, p < 0.001 vs. PL 152.4 [120.1; 307.9] ng/mmol creatinine, p < 0.001). No difference between the groups (p = 0.064).FENa increased (1.08 ± 0.52% after IS and 1.66 ± 1.15% after PL, p = 0.032). ENaC excretion was different within groups (p = 0.019).ConclusionA significantly higher plasma chloride and a lower pH was present in the group receiving isotonic saline. However, u-NGAL and u-KIM-1 increased significantly in both groups after surgery despite absence of changes in creatinine. These results indicate that surgery induced subclinical kidney injury. Also, the IS group had a delayed sodium excretion as compared to the PL group which may indicate that IS affects renal sodium excretion differently from PL.Trial registrationClinicalTrials.gov Identifier: NCT02528448, 19/08/2015

Reduced urinary excretion of neutrophil gelatinase-associated lipocalin as a risk factor for recurrence of febrile urinary tract infection in children.

This study aimed to test the hypothesis that reduced urinary excretions of neutrophil gelatinase-associated lipocalin (NGAL) predispose children to recurrence of febrile urinary tract infection (fUTI). Subjects were 38 children diagnosed with fUTI. To examine risk factors for recurrence of fUTI, the subjects were divided into a non-recurrent group and a recurrent group according to the presence or absence of fUTI over 3 years since the first episode. We measured the urinary NGAL levels in patients with fUTI at the non-infected stage in addition to age-matched healthy control children. In a multiple logistic regression analysis, significant differences between the groups were not observed for age, sex, the prevalence of kidney scarring and bladder bowel dysfunction, urinary β2-microglobulin/creatinine (Cr) level, and serum levels of Cr and Cystatin C, while the recurrent group had significantly more cases with grade III or higher vesicoureteral reflux (p < 0.01). Furthermore, the urinary NGAL/Cr in the recurrent group (median, 3.60 μg/gCr) was significantly lower than that in the non-recurrent group (median, 16.47 μg/gCr; p < 0.01), and age-matched healthy control children (median, 14.14 μg/gCr; p < 0.05). The area under the receiver operating characteristic curve of NGAL/Cr was 0.86 for predicting recurrence of fUTI. A cut-off value of 11.59 μg/gCr had the best accuracy to predict recurrent fUTI yielding a specificity of 78% and a sensitivity of 93%. Reduced levels of urinary NGAL, which protects against urinary infection, are a risk factor for recurrence of fUTI and could serve as a biomarker.

Urinary monocyte chemoattractant protein 1 associated with calcium oxalate crystallization in patients with primary hyperoxaluria

BackgroundPatients with primary hyperoxaluria (PH) often develop kidney stones and chronic kidney disease. Noninvasive urine markers reflective of active kidney injury could be useful to gauge the effectiveness of ongoing treatments.MethodsA panel of biomarkers that reflect different nephron sites and potential mechanisms of injury (clusterin, neutrophil gelatinase-associated lipocalin (NGAL), 8-isoprostane (8IP), monocyte-chemoattractant protein 1(MCP-1), liver-type fatty acid binding protein (L-FABP), heart-type fatty acid binding protein (H-FABP), and osteopontin (OPN)) were measured in 114 urine specimens from 30 PH patients over multiple visits. Generalized estimating equations were used to assess associations between biomarkers and 24 h urine excretions, calculated proximal tubular oxalate concentration (PTOx), and eGFR.ResultsMean (±SD) age at first visit was 19.5 ± 16.6 years with an estimated glomerular filtration rate (eGFR) of 68.4 ± 21.0 ml/min/1.73m2. After adjustment for age, sex, and eGFR, a higher urine MCP-1 concentration and MCP-1/creatinine ratio was positively associated with CaOx supersaturation (SS). Higher urine NGAL and NGAL/creatinine as well as OPN and OPN/creatinine were associated with higher eGFR. 8IP was negatively associated with PTOx and urinary Ox, but positively associated with CaOx SS.ConclusionIn PH patients greater urine MCP-1 and 8IP excretion might reflect ongoing collecting tubule crystallization, while greater NGAL and OPN excretion may reflect preservation of kidney mass and function. CaOx crystals, rather than oxalate ion may mediate oxidative stress in hyperoxaluric conditions. Further studies are warranted to determine whether urine MCP-1 excretion predicts long term outcome or is altered in response to treatment.

The Influence of Salt Loading on the Kidney Injury Biomarker Neutrophil Gelatinase‐Associated Lipocalin in Healthy Young Adults

PurposeThe kidneys play a critical role in blood pressure (BP) regulation. In hypertensive patients and in rodent models, long‐term high dietary sodium (Na+) causes a reduction in kidney function (reduced glomerular filtration rate; GFR). Rodent studies demonstrate that high dietary Na+ increases levels of novel kidney injury biomarkers, independent of changes in BP and GFR. However, it is unclear if short‐term high Na+ increases kidney injury biomarkers in healthy humans. Therefore, we tested the hypothesis that short‐term high Na+ intake increases urinary excretion of the novel kidney injury marker Neutrophil Gelatinase‐Associated Lipocalin (NGAL) in healthy, young adults.MethodsTwenty participants (12 M/8 F; age: 24±4 years; BMI: 23.1±0.6 kg/m2; BP: 112 ±10/64 ±9 mmHg, mean ± SD) participated in a double‐blind, placebo‐controlled, randomized, crossover study. For 10‐days, participants were asked to consume a 2,300 mg/day Na+ diet and supplement with salt (3,900 mg/day of Na+) or placebo (dextrose) capsules. The conditions were separated by at least two weeks and female participants were tested during the placebo phase of their oral contraceptive cycle. Participants collected their urine for the final 24 hours of each supplementation period to assess urine flow rate, and Na+ excretion to verify diet adherence. Brachial BP was measured after at least five minutes of rest. We measured urine and serum creatinine (kinetic modification via the Jaffe reaction) to estimate GFR via creatinine clearance. We used enzyme‐linked immunosorbent assays (Toronto Bioscience) to measure urinary NGAL. Statistical analysis included paired t‐tests and Wilcoxon matched‐pairs signed rank tests.ResultsCompared to placebo, Na+ supplementation increased urinary Na+ excretion (139.9±68.4 vs. 282.5±69.8 mmol/24 hours, p&lt;0.01) but there was no difference in mean arterial BP (77±7 vs. 77±6 mmHg, p=0.64). Serum creatinine concentrations (0.78±0.12 vs. 0.77±0.09 mg/dL, p=0.55) were not different between conditions. However, compared to placebo, Na+ supplementation increased creatinine clearance (110.5±32.9 vs. 145.0±24.9 mL/min, p&lt;0.01) and increased urinary NGAL excretion indexed to urine flow rate (16.9±21.9 vs. 28.4±36.1 ng/min, p=0.02).DiscussionOur preliminary data suggests that salt loading does not alter serum creatinine concentration but increases creatinine clearance and urinary excretion of the kidney injury marker NGAL in healthy young adults.Support or Funding InformationFundingSupported by NIH grants R01HL128388 and K01HL147998, and ACSM Foundation Doctoral Student Research Grant 17‐00521

Urinary Neutrophil Gelatinase-Associated Lipocalin (NGAL) as a Marker of Diabetic Nephropathy in Type 1 Diabetic Patients.

Glomerular and tubulointerstitial damage plays a role in renal function failure in diabetic patients. While both serum and urine levels of neutrophil gelatinase-associated lipocalin (NGAL) show significantly increased levels in acute renal pathologies, the NGAL increase in active phase indicates a reversible condition in chronic cases. 52 type 1 diabetic patients and 30 healthy volunteers participated in the study. The diabetic participants were separated into two groups as follows: a normoalbuminuria group consisting of those with an albumin/creatinine ratio less than 30 mg/g and an albuminuria group consisting of those with an albumin/ creatinine ratio equal or greater than 30 mg/g. Albumin, creatinine and NGAL were measured in all participants. Urinary NGAL median level was 21.1 ng/mL for diabetic patients and 11.9 ng/mL for healthy controls, and the difference between the two groups was statistically significant. When diabetic patients were compared as those with and without albuminuria, the median urinary NGAL levels of normoalbuminuria and albuminuria were 24.7 and 16.1 ng/mL, respectively, but the difference was not statistically significant. Statistically similar results were obtained through evaluation of the ratio of urinary NGAL excretion to creatinine excretion. The NGAL/Cr ratio was significantly higher in diabetic patients than in healthy controls, but no statistically significant difference was found between the diabetic patients with and without albuminuria. Urinary NGAL excretion in type 1 diabetic patients is found to be increased over a wide range, but it does not correlate with urinary albumin excretion. In this regard, urinary NGAL excretion should not be used as an alternative to microalbuminuria in detecting diabetic nephropathy. The greater amount of NGAL excretion among diabetic patients may be due to diabetic nephropathy with possible tubulointerstitial damage pathologies.

Assessment of renal function in obese and overweight children with NGAL and KIM-1 biomarkers.

Aim and background: the incidence of obesity has increased among children, and obesity has been considered an independent risk factor for chronic kidney disease. We aimed to determine the degree of kidney function impairment by evaluating urine neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) levels. Materials and methods: in total, 15 obese, 26 overweight, and 26 control adolescents aged 10 to 16 years were enrolled into the study. Urine samples were evaluated for NGAL and KIM-1 levels using enzyme-linked immunosorbent assay kits. We investigated the association between obesity and related comorbidities with urinary NGAL and KIM-1 excretion. Results: no significant differences were noted between the obese, overweight, and control groups in urinary NGAL and KIM-1 excretion (p = 0.327 and p = 0.917, respectively). In the obese and overweight groups urinary NGAL levels were 50.39 [30.88-74.22] in females and 26.67 [23.24-45.59] in males (p = 0.013). Also, urinary NGAL levels were increased in obese and overweight adolescents with LDL dyslipidemia at 64.12 [30.98-114.32] as compared to those without LDL dyslipidemia: 39.51 [25.59.56.37] (p = 0.024). Furthermore, a correlation was observed between insulin and homeostasis model assessment of insulin resistance levels with the NGAL/creatinine ratio in the overweight group (r = 0.515; p = 0.008, and r = 0.483; p = 0.014, respectively). Such correlation was not found in the obese group. Conclusion: the effect of obesity on renal function could not be determined in children. A longer exposure may be required for obesity-induced disruption of renal function in children. Renal function may be disrupted by dyslipidemia in obese adolescents. Furthermore, obesity impaired renal function in female adolescents. The normalization of these urinary markers as related to urine creatinine should be discussed.

Effect of endoplasmic reticulum stress-mediated excessive autophagy on apoptosis and formation of kidney stones

AimsThis study was designed to reveal the role and underlying mechanism of excessive autophagy mediated by ERS via the PERK-eIF2α pathway in the apoptosis and formation of CaOx kidney stones. Main methodsEthylene glycol (EG) was used to establish a rat model of CaOx kidney stones, and 100 mg/kg of ERS inhibitor 4-phenylbutyric acid (4-PBA) or 60 mg/kg of autophagy inhibitor chloroquine (CQ) was administered daily to the rats. Four weeks after administration, we collected blood and kidney tissues to analyze the occurrence of ERS and autophagy, apoptosis, renal function, renal tubular crystal deposition, and kidney damage, respectively. Key findingsWe observed that both 4-PBA and CQ treatment significantly inhibited the excessive autophagy and reduced apoptosis as well as decreasing p-PERK and p-eIF2α expressions. Meanwhile, the proportion of kidney weight, contents of creatinine and blood urea nitrogen, excretion of neutrophil gelatinase-associated lipocalin and kidney injury molecule 1, and renal tubular deposition were markedly down-regulated. SignificanceThe findings in this study suggested that ERS induced excessive autophagy via the PERK-eIF2α pathway, regulating cell damage and apoptosis. ERS-mediated inhibition of excessive autophagy effectively protected kidney function and prevented the apoptosis and formation of kidney stones.

Influence of renal ischaemia‐reperfusion injury on renal neutrophil gelatinase‐associated lipocalin receptor (24p3R) in rats

The neutrophil gelatinase-associated lipocalin (NGAL) receptor (24p3R) is expressed in distal nephron and contributes to the endocytosis of NGAL in urine. This study was undertaken to evaluate an influence of renal ischaemia-reperfusion injury on 24p3R. Unilateral renal pedicle was clamped for 0, 10, 20, 30, or 45minutes in male Wistar rats. Urine was collected for 24hours after reperfusion, and ischaemic kidney and blood sample were obtained. Apparent histological injury in the ischaemic kidney was detected in the 30 and 45minutes-treated groups. Urinary NGAL excretion elevated in rats with renal ischaemia for more than 20minutes, while serum creatinine increased in rats for more than 30minutes of ischaemia. Renal protein expression of NGAL did not significantly change. Renal mRNA expressions of megalin and cubilin, which are expressed at renal proximal tubules and uptake NGAL, decreased in animals with renal ischaemia for more than 20minutes. Renal protein expression of 24p3R, which is expressed at renal distal tubules and uptake NGAL, decreased in rats with renal ischaemia for 45min. This study showed for the first time that renal 24p3R decreased in response to renal ischaemia. As relatively longer renal ischaemia (45minutes) decreased renal 24p3R protein and increased urinary NGAL excretion, the down-regulation of 24p3R protein might contribute to the elevated urinary excretion of NGAL in rats with unilateral ischaemia-reperfusion injury.

Increased Intra-abdominal Pressure Induces Acute Kidney Injury in an Experimental Model of Congestive Heart Failure

Congestive heart failure (CHF) entails a complex interaction between the heart and the kidney that represents a clinical entity called cardiorenal syndrome (CRS). One of the mechanisms underlying CRS includes increased intra-abdominal pressure (IAP). We examined the effect of elevated IAP on kidney function in rats with low- and high-output CHF. Rats with compensated and decompensated CHF induced by means of aortocaval fistula, rats with myocardial infraction (MI) induced by means of left anterior descending artery ligation, and sham control rats were subjected to either 10 or 14 mm Hg IAP. Urine flow (V), Na+ excretion (UNaV), glomerular filtration rate (GFR), and renal plasma flow (RPF) were determined. The effects of pretreatment with tadalafil (10 mg/kg orally for 4 days) on the adverse renal effects of IAP were examined in decompensated CHF and MI. Basal V and GFR were significantly lower in rats with decompensated CHF compared with sham control rats. Decompensated CHF rats and MI rats subjected to 10 and 14 mm Hg IAP exhibited more significant declines in V, UNaV, GFR and RPF than compensated and sham controls. Elevated IAP also induced tubular injury, as evidenced by significantly increased absolute urinary excretion of neutrophil gelatinase-associated lipocalin. In addition, in a nonquantitative histologic analysis, elevated IAP was associated with increase in necrosis and cell shedding to the tubule lumens, especially in the decompensated CHF subgroup. Pretreatment of decompensated CHF rats and MI rats with tadalafil ameliorated the adverse renal effects of high IAP. Elevated IAP contributes to kidney dysfunction in high- and low-cardiac output CHF. IAP induces both hemodynamic alterations and renal tubular dysfunction. These deleterious effects are potentially reversible and can be ameliorated with the use of phosphodiesterase-5 inhibition.

Protective Role of Apelin Against Cyclosporine-Induced Renal Tubular Injury in Rats

BackgroundCyclosporine (CsA) usually reduces glomerular filtration rate (GFR) but also can induce tubular injury without resulting in GFR reduction. Apelin is an endogenous ligand for the apelin receptor and has diverse physiologic roles related to hemodynamic or metabolic processes. We investigated the renoprotective role of apelin against CsA-induced tubular toxicity in rats. MethodsRats were given CsA (15 mg/kg/day) and/or apelin-13 (15 μg/kg/day) for 7 days via subcutaneous injection. We performed serum and urinary assays of creatinine and neutrophil gelatinase-associated lipocalin (NGAL) to estimate renal injury and performed Western blotting for endothelial nitric oxide synthase and nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) to document the underlying mechanism. ResultsThe CsA-treated group showed increased urinary creatinine excretion, polyuria, and renal glycosuria without GFR reduction, suggesting adequate CsA-induced renal tubular injury. Urinary NGAL excretion also increased significantly in the CsA group. Conversely, apelin attenuated CsA-induced tubular injury and had no effect on urinary NGAL excretion. In histopathologic examination, the apelin-treated group had lower tubulo-interstitial injury scores compared with those in the CsA group. Regarding the effects of apelin, our results indicate that apelin provides protection against CsA-induced tubular injury by activating nitric oxide and/or the NFATc1 pathway. Notably, we also found that CsA inhibits renal glucose reabsorption by reducing Na+-K+ ATPase expression and that apelin reverses reduced renal glucose reabsorption by CsA in tubular cells. ConclusionsOur study demonstrates the renoprotective effect of apelin against CsA-induced renal tubular toxicity and provides novel insights into the effects of CsA and apelin on renal tubular cells.

Identification of a sensitive urinary biomarker, selenium-binding protein 1, for early detection of acute kidney injury

ABSTRACTAcute kidney injury (AKI) is associated with increased mortality rate in patients but clinically available biomarkers for disease detection are currently not available. Recently, a new biomarker, selenium-binding protein 1 (SBP1), was identified for detection of nephrotoxicity using proteomic analysis. The aim of this study was to assess the sensitivity of urinary SBP1 levels as an early detection of AKI using animal models such as cisplatin or ischemia/reperfusion (I/R). Sprague–Dawley rats were injected with cisplatin (6 mg/kg, once i.p.) and sacrificed at 1, 3, or 5 days after treatment. Ischemia was achieved by bilaterally occluding both kidneys with a microvascular clamp for 45 min and verified visually by a change in tissue color. After post-reperfusion, urine samples were collected at 9, 24, and 48 hr intervals. Urinary excretion of protein-based biomarkers was measured by Western blot analysis. In cisplatin-treated rats, mild histopathologic alterations were noted at day 1 which became severe at day 3. Blood urea nitrogen (BUN) and serum creatinine (SCr) levels were significantly increased at day 3. Levels of urinary excretion of SBP1, neutrophil gelatinase-associated lipocalin (NGAL), and a tissue inhibitor of metalloproteinase-1 (TIMP-1) were markedly elevated at day 3 and 5 following drug treatment. In the vehicle-treated I/R group, serum levels of BUN and SCr and AST activity were significantly increased compared to sham. Urinary excretion of SBP1 and NGAL rose markedly following I/R. The urinary levels of SBP1, NGAL, TIMP-1, and KIM-1 proteins excreted by AKI patients and normal subjects were compared. Among these proteins, a marked rise in SBP1 was observed in urine of patients with AKI compared to normal subjects. Based upon receiver-operator curves (ROC), SBP1 displayed a higher area under the curve (AUC) scores than levels of SCr, BUN, total protein, and glucose. In particular, SBP1 protein was readily detected in small amounts of urine without purification. Data thus indicate that urinary excretion of SBP1 may be useful as a reliable biomarker for early diagnosis of AKI in patients.

Testing Danegaptide Effects on Kidney Function after Ischemia/Reperfusion Injury in a New Porcine Two Week Model.

IntroductionIschemia/reperfusion injury (I/R-I) is a leading cause of acute kidney injury (AKI) and is associated with increased mortality. Danegaptide is a selective modifier of the gap junction protein connexion 43. It has cytoprotective as well as anti-arrhythmic properties and has been shown to reduce the size of myocardial infarct in pigs. The aim of this study was to investigate the ischemia-protective effect of Danegaptide in a porcine renal I/R-I model with two weeks follow up.MethodsUnilateral renal I/R-I was induced in pigs by clamping the left renal artery over a two hour period. The model allowed examination of renal blood flow by magnetic resonance imaging (MRI) and the measurement of single kidney GFR two weeks after injury. Eleven animals were randomized to Danegaptide-infusion while nine animals received placebo. Kidney histology and urinary neutrophil gelatinase-associated lipocalin (NGAL) excretion were included as markers of AKI.ResultsUnilateral kidney I/R-I resulted in an immediate ~50% GFR reduction, associated with a four-fold increase in urinary NGAL-excretion. Fourteen days after I/R-I, the total GFR was ~75% of baseline with a significantly lower GFR in the injured left kidney compared to the right kidney. No differences in GFR were observed between the treated and non-treated animals immediately after I/R-I or at Day 14. Furthermore, no differences were observed in the urinary excretion of NGAL, renal blood flow or other markers of renal function.ConclusionsAs expected this porcine renal I/R-I model was associated with reduced GFR two weeks after injury. Danegaptide did not improve renal function after I/R-I.

Growth arrest specific 2-like protein 1 expression is upregulated in podocytes through advanced glycation end-products.

Growth arrest specific 2-like protein 1 (GAS2L1) protein is a member of the GAS2 family of proteins, known to regulate apoptosis and cellular cytoskeleton reorganization in different cells. Recently we identified that Gas2l1 gene expression in podocytes is influenced by advanced glycation end product-bovine serum albumin(AGE-BSA). The study was performed employing cultured podocytes and diabetic ( db/db ) mice, a model of type 2 diabetes. Akbuminuria as wellas urinary neutrophil gelatinase-associated lipocalin (NGAL) excretion as measured with specific ELISAs. Gene expression was analysed via semiquantitative and real-time polymerase chain reaction. The protein levels were determined by western blotting and immunostaining. We found that the Gas2l1 α isoform is expressed in podocytes. Treatment with AGE-BSA induced Gas2l1 α and Gas2 mRNA levels compared with controls incubated with non-glycated control BSA (Co-BSA). Moreover, application of the recombinant soluble receptor of AGEs (sRAGE), a competitor of cellular RAGE, reversed the AGE-BSA effect. Interestingly, AGE-BSA also increased the protein levels of GAS2L1α in a RAGE-dependent manner, but did not affect the GAS2 expression. Periodic acid-Schiff staining and albuminuria as well as urinary NGAL excretion revealed that db/db mice progressively developed diabetic nephropathy with renal accumulation of N ε -carboxy-methyl-lysine (immunohistochemistry, western blots). Analyses of GAS2L1α and GAS2 proteins in diabetic mice revealed that both were significantly elevated relative to their non-diabetic littermates. In addition, GAS2L1α and GAS2 proteins positively correlated with the accumulation of AGEs in the blood plasma of diabetic mice and the administration of sRAGE in diabetic mice reduced the glomerular expression of both proteins. We show for the first time that the protein expression of GAS2L1α in vitro and in vivo is regulated by the AGE-RAGE axis. The suppression of AGE ligation with their RAGE in diabetic mice with progressive nephropathy reversed the GAS2L1α expression, thus suggesting a role of GAS2L1α in the development of diabetic disease, which needs to be further elucidated.

Independent associations of urine neutrophil gelatinase&amp;ndash;associated lipocalin and serum uric acid with interstitial fibrosis and tubular atrophy in primary glomerulonephritis

The degree of interstitial fibrosis and tubular atrophy (IFTA) is one of the strongest prognostic factors in glomerulonephritis (GN). In experimental models, high serum uric acid (UA) could contribute to IFTA through direct effects on the renal tubules, but the significance of this process has not been evaluated in patients. Urine neutrophil gelatinase–associated lipocalin (NGAL) is produced by renal tubules following acute or chronic damage. We investigated the relationship between UA and NGAL excretion in primary GN and tested whether these biomarkers are independently associated with IFTA. Urine and blood were collected from patients on the day of kidney biopsy. IFTA was assessed semi-quantitatively. Fifty-one patients with primary GN were enrolled. NGAL/creatinine correlated significantly with proteinuria but not with glomerular filtration rate (GFR). By contrast, UA correlated with GFR but not with proteinuria. NGAL/creatinine did not correlate with UA. Both NGAL/creatinine and UA increased with the severity of IFTA. By multivariate analysis, GFR, NGAL/creatinine, and UA were independently associated with moderate-to-severe IFTA. Combining UA and NGAL/creatinine with classical predictors (proteinuria and GFR) tended to improve discrimination for moderate-to-severe IFTA. Findings that UA was unrelated to urinary NGAL excretion suggest that the two biomarkers reflect different pathways related to the development of IFTA in primary GN. Both NGAL/creatinine and UA were independently associated with moderate-to-severe IFTA.

Mild systemic thermal therapy ameliorates renal dysfunction in a rodent model of chronic kidney disease.

Thermal therapy has become a nonpharmacological therapy in clinical settings, especially for cardiovascular diseases. However, the practical role of thermal therapy on chronic kidney disease remains elusive. We performed the present study to investigate whether a modified thermal protocol, repeated mild thermal stimulation (MTS), could affect renal damages in chronic kidney disease using a mouse renal ablation model. Mice were subjected to MTS or room temperature (RT) treatment once daily for 4 wk after subtotal nephrectomy (Nx) or sham operation (Sh). We revealed that MTS alleviated renal impairment as indicated by serum creatinine and albuminuria in Nx groups. In addition, the Nx + MTS group showed attenuated tubular histological changes and reduced urinary neutrophil gelatinase-associated lipocalin excretion approximately by half compared with the Nx + RT group. Increased apoptotic signaling, such as TUNEL-positive cell count and cleavage of caspase 3, as well as enhanced oxidative stress were significantly reduced in the Nx + MTS group compared with the Nx + RT group. These changes were accompanied with the restoration of kidney Mn-SOD levels by MTS. Heat shock protein 27, a key molecular chaperone, was phosphorylated by MTS only in Nx kidneys rather than in Sh kidneys. MTS also tended to increase the phosphorylation of p38 MAPK and Akt in Nx kidneys, possibly associated with the activation of heat shock protein 27. Taken together, these results suggest that modified MTS can protect against renal injury in a rodent model of chronic kidney disease.