Serum soluble α-Klotho levels in patients with diabetic nephropathy.

BACKGROUND: The study aimed to determine the variability in the stages of diabetic nephropathy by examining specific biochemical functions associated with the target organ. As a result, various biochemical parameters were assessed in all of the groups under investigation. MATERIAL AND METHODS: These parameters encompassed soluble α-Klotho and serum insulin, which were determined through ELISA. Additionally, spectrophotometric methods were employed to assess other parameters such as blood levels of urea in all groups. Instead of using HPLC method, HbA1c levels were determined. Blood and urine samples were obtained from a total of 90 participants, who were aged between 37 and 70 years. A total of 70 patients were categorized into three groups according to their ACR. The first group consisted of patients with an ACR value of less than 30 mg/g. The second group included patients with an ACR value ranging from 30 to 300 mg/g. The third group comprised patients with an ACR value greater than 300 mg. Additionally, the study also involved 20 healthy individuals. RESULT: The serum soluble α-Klotho in the patient group was significantly lower than that of the healthy subjects. There were strong negative correlations between serum soluble α-Klotho and both ACR and HOMA-IR. The AUC value was excellent, measuring at 0.93 with a p < 0.0001. CONCLUSIONS: Soluble α-Klotho levels in the sera of diabetic patients were shown to be lower and significantly linked to patients with diabetic nephropathy. This implies that klotho levels may be influenced by ACR in addition to playing a significant role in insulin resistance.

Urinary Klotho Excretion: A Key Regulator of Sodium Homeostasis in Chronic Kidney Disease Stage 2-4.

BACKGROUND Soluble alpha-klotho (klotho) is considered an important regulator of mineral homeostasis in patients with chronic kidney disease (CKD). Since the mineral transport proteins are located on the apical membrane of renal tubular cells, we hypothesized that urine klotho may also be involved in their homeostasis. We aimed to investigate the associations between serum and urine klotho and their impacts on mineral homeostasis in patients with stage 2 to 4 CKD. MATERIAL AND METHODS Serum, spot urine, and 24-h urine of klotho were measured by using enzyme-linked immunosorbent assay. Fractional excretion of sodium, potassium, calcium, phosphate, magnesium, and klotho were calculated. RESULTS A total of 53 patients with CKD stages 2 to 4 were enrolled in this cross-sectional study. The mean age was 71.1±10.5 years, and 68% were men. Linear regression analysis showed that serum log-transformed klotho was negatively associated with log-transformed fractional excretion of klotho (log-FEKlotho) (ß=-0.085, P=0.02), showing that urinary klotho excretion could negatively regulate serum klotho levels. Moreover, our multivariate stepwise regression showed log-fractional excretion of sodium was positively associated with log-FEKlotho (ß=0.138, P=0.032). This implied urinary klotho excretion positively regulated urinary sodium excretion. CONCLUSIONS Our study showed that urine klotho excretion resulted in decreased serum klotho levels and enhanced urinary sodium excretion in patients with CKD stages 2 to 4. In addition to serum klotho, we found, for the first time, that urine klotho also played a significant role in sodium homeostasis.

Investigation on urinary and serum alpha klotho in dogs with chronic kidney disease.

BACKGROUND: As a co-receptor for fibroblast growth factor 23, klotho plays a pivotal role in phosphate metabolism. The kidney is known to be the main source of soluble alpha-klotho and the principal regulator of its concentration. Previous studies in human participants showed that the concentration of soluble alpha-klotho in serum and urine decreased in chronic kidney disease (CKD) patients. However, no previous study has assessed soluble alpha-klotho levels in dogs. This study aimed to measure serum and urinary alpha-klotho levels in CKD dogs and identify their associations with International Renal Interest Society (IRIS) CKD stages and other parameters known to be associated with CKD. RESULTS: Serum and urinary alpha klotho concentrations were measured by a commercially available canine-specific sandwich enzyme-linked immunosorbent assay kit and compared between groups by a nonparametric Kruskal-Wallis test. Spearman's correlation coefficient was used to evaluate the relationships between variables. A stepwise multiple regression analysis was performed to estimate the effects of independent predictors on klotho concentrations. The urine klotho-to-creatinine ratio (UrKl/Cr) was significantly lower in stage 3 dogs than the control group and was significantly lower in dogs with stage 3 and 4 CKD than in those with stage 1 and 2 disease. UrKl/Cr was negatively correlated with serum symmetric dimethylarginine (sSDMA), blood urea nitrogen (BUN), creatinine, and phosphorus concentration. Serum alpha-klotho concentration in dogs with stages 2 and 3 CKD was significantly lower than those in the control group. There was no significant correlation between serum alpha-klotho and BUN, creatinine, and phosphorus concentrations. No statistically significant differences were observed in UrKl/Cr and serum alpha-klotho concentration between groups based on sex, age, urine protein-to-creatinine ratio (UPC), or blood pressure. CONCLUSIONS: UrKl/Cr decreased in dogs with advanced CKD, and it was negatively correlated with sSDMA, BUN, creatinine, and phosphorus concentrations. Thus, klotho is associated with CKD and its clinical consequences, including CKD-mineral bone disorder, in dogs. Although serum klotho concentration was negatively correlated with sSDMA levels, it was not apparently related to IRIS CKD stage or other parameters known to be associated with CKD.

Lower urinary α-Klotho is associated with lower angiotensin-(1-7) and higher blood pressure in young adults born preterm with very low birthweight.

Early-life factors including preterm birth and VLBW increase the risk of hypertension, but the mechanisms remain poorly understood. Reductions in the anti-aging protein α-klotho are associated with hypertension, possibly due to angiotensin (Ang) II activation, but the mechanisms are incompletely understood and clinical evidence is lacking. The association of α-klotho with the alternative Ang-(1-7) pathway, which counteracts Ang II to lower BP, is undescribed. We hypothesized that lower urinary α-klotho is associated with higher BP and lower urinary Ang-(1-7) in preterm-born VLBW young adults. In a cross-sectional analysis of data from a prospective cohort of 141 preterm-born VLBW young adults, we assessed the associations among urinary α-klotho/creatinine, Ang II/creatinine, Ang-(1-7)/creatinine, Ang II/Ang-(1-7), and BP using generalized linear models adjusted for age and hypertensive pregnancy and conducted a sensitivity analysis in 32 term-born young adults. Among those born preterm, lower α-klotho/creatinine was associated with higher systolic BP (adjusted ß (aß): -2.58 mm Hg, 95% CI -4.99 to -0.17), lower Ang-(1-7)/creatinine (ln aß: 0.1, 0.04-0.16), and higher Ang II/Ang-(1-7) (ln aß: -0.14, -0.21 to -0.07). In term-born participants, α-klotho/creatinine was inversely associated with Ang II/creatinine (ln aß: -0.15, -0.27 to -0.03) and Ang II/Ang-(1-7) (ln aß: -0.15, -0.27 to -0.03). In preterm-born young adults with VLBW, lower urinary α-klotho/creatinine was associated with higher SBP, lower urinary Ang-(1-7)/creatinine, and higher urinary Ang II/Ang-(1-7). Reduced renal α-klotho expression could lead to renal Ang-(1-7) suppression as a novel mechanism for the development of hypertension among individuals born preterm with VLBW.

First Report of a Patient with MPS Type VII, Due to Novel Mutations in GUSB, Who Underwent Enzyme Replacement and Then Hematopoietic Stem Cell Transplantation.

We report the case of a boy who was diagnosed with mucopolysaccharidosis (MPS) VII at two weeks of age. He harbored three missense ß-glucuronidase (GUSB) variations in exon 3: two novel, c.422A>C and c.424C>T, inherited from his mother, and the rather common c.526C>T, inherited from his father. Expression of these variations in transfected HEK293T cells demonstrated that the double mutation c.422A>C;424C>T reduces ß-glucuronidase enzyme activity. Enzyme replacement therapy (ERT), using UX003 (vestronidase alfa), was started at four months of age, followed by a hematopoietic stem cell allograft transplantation (HSCT) at 13 months of age. ERT was well tolerated and attenuated visceromegaly and skin infiltration. After a severe skin and gut graft-versus-host disease, ERT was stopped six months after HSCT. The last follow-up examination (at the age of four years) revealed a normal psychomotor development, stabilized growth curve, no hepatosplenomegaly, and no other organ involvement. Intriguingly, enzyme activity had normalized in leukocytes but remained low in plasma. This case report illustrates: (i) The need for an early diagnosis of MPS, and (ii) the possible benefit of a very early enzymatic and/or cellular therapy in this rare form of lysosomal storage disease.

Urine klotho is a potential early biomarker for acute kidney injury and associated with poor renal outcome after cardiac surgery.

BACKGROUND: Current paradigms of detecting acute kidney injury (AKI) are insensitive and non-specific. Klotho is a pleiotropic protein that is predominantly expressed in renal tubules. In this study, we evaluated the diagnostic and prognostic roles of urine Klotho for AKI following cardiac surgery. METHODS: We conducted a prospective study involving 91 patients undergoing cardiac surgery. AKI was defined according to the AKIN definition. The renal outcomes within 7 days after operation were evaluated. Perioperative levels of urine Klotho and urine neutrophil gelatinase-associated lipocalin (NGAL) were measured by using ELISA. RESULTS: Of 91 participants, 33 patients (36.26%) developed AKI. Of these AKI patients, 21 (63.64%), 8 (24.24%), and 4 (12.12%) were staged 1, 2, and 3, respectively. Serum creatinine in AKI patients began to slightly increase at first postoperative time and reached the AKI diagnostic value 1 day after operation. Postoperative urine Klotho peaked at the first postoperative time (0 h after admission to the intensive care unit (ICU)) in patients with AKI, and was higher than that in non-AKI patients up to day 3. The AUC of detecting AKI for urine Klotho was higher than urine NGAL at the first postoperative time and 4 h after admission to the ICU. In a multivariate model, increased first postoperative urine Klotho may be an independent predictor for AKI occurrence following cardiac surgery. The concentrations of first postoperative urine Klotho were higher in AKI stage 2 and 3 than those in stage 1 (p < 0.05), and were higher in patients with incomplete recovery of renal function than those with complete recovery (p < 0.05). CONCLUSIONS: Urine Klotho may serve as an early biomarker for AKI and subsequent poor short-term renal outcome in patients undergoing cardiac surgery.

Bicarbonate Supplement Restores Urinary Klotho Excretion in Chronic Kidney Disease: A Pilot Study.

OBJECTIVE: We tested the hypothesis that correcting acidosis may improve urinary Klotho excretion and serum α-Klotho. DESIGN: This is a prospective, interventional, nonrandomized, open-label trial study. In this study setting, metabolic acidosis is commonly observed during chronic kidney disease (CKD). We reported a positive relationship between serum bicarbonate (Sbicar) and serum α-Klotho in these patients. SUBJECTS: The study involved 20 patients with a known kidney disease referred for renal checkup. Inclusion criteria were age ≥ 18 years, CKD stage 3-5 non dialysis, Sbicar < 22 mmol/L, and not receiving bicarbonate supplementation. INTERVENTION: Patients were then prescribed 1 g of oral sodium bicarbonate 3 times per day for 4 weeks. MAIN OUTCOME MEASURE: Patients were evaluated at two and 4 weeks by blood and urine measurements. RESULTS: Mean serum Klotho was 615 ± 287 pg/mL, and mean serum Sbicar was 19.3 ± 1.7 mmol/L at baseline. Sbicar increased from baseline at two (23.9 ± 2.9 mmol/L, P < .001) and 4 weeks (23.4 ± 1.9 mmol/L, P < .001). There was no change in serum Klotho at two (630 ± 333 mmol/L) and 4 weeks (632 ± 285 mmol/L). By contrast, urine Klotho/creatinine ratio, which was very low at baseline (34.6 ± 31.6 pg/mmoL), increased by 320% at two weeks (P < .005) and by 280% at 4 weeks (P < .01). CONCLUSIONS: Correcting acidosis by oral administration of sodium bicarbonate rapidly increases the urine excretion of soluble α-Klotho in CKD patients. However, a 4-week bicarbonate treatment was not able to increase serum α-Klotho. A longer study may confirm this pilot observation and increase serum Klotho, which has been shown to exert a protective cardiovascular effect during CKD.

Effects of Pentoxifylline on Soluble Klotho Concentrations and Renal Tubular Cell Expression in Diabetic Kidney Disease.

OBJECTIVE: The effect of pentoxifylline on Klotho levels in patients with type 2 diabetes mellitus with chronic kidney disease (CKD) was assessed in a post hoc analysis of the Pentoxifylline for Renoprotection in Diabetic Nephropathy (PREDIAN) trial. RESEARCH DESIGN AND METHODS: Circulating and urinary tumor necrosis factor-α (TNF-α) and Klotho were measured before and after 1 year of pentoxifylline. The effect on Klotho expression was assessed in cultured renal tubular cells. RESULTS: Pentoxifylline administration resulted in decreased serum and urinary TNF-α, whereas serum and urinary Klotho increased significantly. Changes in urinary Klotho, urinary TNF-α, and phosphorus were associated with changes in serum Klotho; changes in estimated glomerular filtration rate, urinary TNF-α, and albuminuria were related to urinary Klotho variation. In renal tubular cells, pentoxifylline prevented the decrease in Klotho expression induced by inflammatory cytokines or albumin. CONCLUSIONS: Pentoxifylline increased Klotho levels in patients with diabetes with stage 3-4 CKD and prevented reduced Klotho expression in vitro. This beneficial effect may be related to anti-inflammatory and antialbuminuric activity.

Ubiquitin COOH-terminal hydrolase L1 deletion is associated with urinary α-klotho deficiency and perturbed phosphate homeostasis.

Loss of ubiquitin COOH-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme required for neuronal function, led to hyperphosphatemia accompanied by phosphaturia in mice, while calcium homeostasis remained intact. We therefore investigated the mechanisms underlying the phosphate imbalance in Uchl1-/- mice. Interestingly, phosphaturia was not a result of lower renal brush border membrane sodium-phosphate cotransporter expression as sodium-phosphate cotransporter 2a and 2c expression levels was similar to wild-type levels. Plasma parathyroid hormone and fibroblast growth factor 23 levels were not different; however, fibroblast growth factor 23 mRNA levels were significantly increased in femur homogenates from Uchl1-/- mice. Full-length and soluble α-klotho levels were comparable in kidneys from wild-type and Uchl1-/- mice; however, soluble α-klotho was reduced in Uchl1-/- mice urine. Consistent with unchanged components of 1,25(OH)2D3 metabolism (i.e., CYP27B1 and CYP24A1), sodium-phosphate cotransporter 2b protein levels were not different in ileum brush borders from Uchl1-/- mice, suggesting that the intestine is not the source of hyperphosphatemia. Nonetheless, when Uchl1-/- mice were fed a low-phosphate diet, plasma phosphate, urinary phosphate, and fractional excretion of phosphate were significantly attenuated and comparable to levels of low-phosphate diet-fed wild-type mice. Our findings demonstrate that Uchl1-deleted mice exhibit perturbed phosphate homeostasis, likely consequent to decreased urinary soluble α-klotho, which can be rescued with a low-phosphate diet. Uchl1-/- mice may provide a useful mouse model to study mild perturbations in phosphate homeostasis.

Heparanase expression in blood is sensitive to monitor response to anticancer treatment in pancreatic cancer, a pilot study.

BACKGROUND: /Objectives: High heparanase level was shown in maliganant tumor; however, whether or not heparanase may serve as a sensitive marker to monitor response to anticancer treatment is still unknown. METHODS: In the pilot study, heparanase mRNA expression in peripheral blood mononuclear cell fraction (PBMC) and activity in plasma and urine were detected by quantitative real time RT-PCR and heparan-degrading enzyme assay in 31 pancreatic cancer patients. RESULTS: Heparanase mRNA and activity in samples from cancer patients were significantly higher than that in healthy donors. Both heparanase mRNA and activity in plasma and urine decreased significantly in 17 patients who underwent R0 resection, but increased remarkably in 6 patients when recurrence or metastasis occurred (P < 0.05). However, those who underwent R1 or R2 resection in 6 patients kept stable. For 8 patients who received chemotherapy, heparanase mRNA and activity in plasma and urine decreased in each of the samples (P < 0.05). Patients with high heparanase mRNA (≥a cutoff value of 1.84) in PBMC and activity in plasma (≥1.30U/ml) were associated with a poor postoperative survival (P = 0.02 and P = 0.04). CONCLUSIONS: Heparanase mRNA in PBMC and activity in plasma are closely correlated with therapeutic responsiveness and survival time, indicating that heparanase level in blood might be a sensitive but non-specific marker to monitor patients' response to anticancer treatment and to predict survival.

Increased Granulocyte Heparanase Activity in Neutrophils from Patients with Lupus Nephritis and Idiopathic Membranous Nephropathy.

Heparanase is a ß-glucuronidase that cleaves sugar chains of heparan sulfate proteoglycans. It is believed that heparanase may be involved in the pathogenesis of proteinuria. The aim of this study was to assess the significance of heparanase in the pathogenesis of particular glomerulonephritis types. The evaluation of heparanase activity in serum, urine, and granulocytes and superoxide dismutase (SOD) activity in granulocytes of patients with lupus nephritis (n = 17), membranous nephropathy (n = 11), IgA nephropathy (n = 12), focal and segmental glomerulosclerosis (n = 18), mesangiocapillary glomerulonephritis (n = 12) and in 19 healthy volunteers were performed. The heparanase activity in granulocytes of patients with lupus nephritis and membranous nephropathy was higher than heparanase activity in granulocytes in the control group (p = 0.02 in both cases). This is the first observation of this phenomenon. There was no difference between SOD activity in granulocytes of patients with all assessed types of glomerulonephritis and the control group. A positive correlation between heparanase activity in urine and double-strain DNA antibodies (r = 0.51; p = 0.04), and reverse correlations between heparanase in urine and hemolytic activity of the complement (r = -0.57; p = 0.03) in the lupus nephritis group, and between heparanase activity in granulocytes and serum total protein level (r = -0.69; p = 0.02) in membranous nephropathy were observed. Increase in heparanase activity without changes in superoxide dismutase activity in the granulocytes from patients with lupus nephritis and membranous nephropathy was observed. It may be used as one of the markers of these disease activities.

Variations in enzymatic hydrolysis efficiencies for amitriptyline and cyclobenzaprine in urine.

A collaborative study was conducted to investigate discrepancies in recoveries of two commonly prescribed compounds, amitriptyline and cyclobenzaprine, in patient urine samples when hydrolyzed with different enzymes from different sources. A 2- to 10-fold increase in analyte recoveries was seen for patient samples hydrolyzed using a recombinant ß-glucuronidase (IMCSzyme™) over samples hydrolyzed with ß-glucuronidase from Haliotis rufescens We report outcomes from four commercially available ß-glucuronidase enzymes (IMCSzyme™, Patella vulgata, Helix pomatia and H. rufescens) on patient samples that tested positive for amitriptyline and cyclobenzaprine. Our results confirm reduced hydrolysis of glucuronides by ß-glucuronidases isolated from mollusks, but near complete conversion when using the recombinant enzyme. Our premise is that systematic differences in hydrolysis efficiencies due to varying substrate affinity among enzyme subtypes potentially impacts accuracy and reliability of measurements.

Decreased plasma α-Klotho predict progression of nephropathy with type 2 diabetic patients.

AIM: The potential role of soluble α-klotho in diabetic kidney disease has not yet been evaluated. The aim of this study was to evaluate the association of plasma and/or urine α-klotho with the progression of type 2 diabetic nephropathy. METHODS: The baseline values of plasma and urine α-klotho were measured in 147 patients with type 2 diabetes mellitus with an estimated glomerular filtration rate (eGFR) of ≥60mL/min/1.73m(2). In this prospective observational study, a total of 109 type 2 diabetic patients were followed up for 34months (8-50 months). RESULTS: Plasma α-klotho, but not urine α-klotho, was negatively correlated with the decline of eGFR (r=-0.304, P=0.001; r=0.042, P=0.068, respectively). After adjusting for several clinical parameters, baseline eGFR and urine ACR, plasma α-klotho was significantly associated with the decline of eGFR (r=-0.219, P=0.008). In the normoalbuminuria group (n=63), the plasma α-klotho remained significantly associated with a decline in eGFR (r=0.324, P=0.004) in the final model. CONCLUSIONS: It is suggested that plasma α-klotho may be an early biomarker for predicting renal impairment in type 2 diabetic patients. The disappearance of a compensatory increase of plasma α-klotho might be a predictive marker for the progression of type 2 diabetic nephropathy.

Klotho and S100A8/A9 as Discriminative Markers between Pre-Renal and Intrinsic Acute Kidney Injury.

Early detection and accurate differentiation of the cause of AKI may improve the prognosis of the patient. However, to date, there are few reliable biomarkers that can discriminate between pre-renal and intrinsic AKI. In this study, we determined whether AKI is associated with altered serum and urinary levels of Klotho, S100A8/A9 (an endogenous ligand of toll-like receptor 4), and neutrophil gelatinase-associated lipocalin (NGAL), which may allow differentiation between pre-renal and intrinsic AKI. A volume-depleted pre-renal AKI model was induced in male Sprague Dawley rats fed a low-salt diet (0.03%) without water 96 h before two intraperitoneal (IP) injections of furosemide (20 mg/kg) at a 24 h interval. In contrast, in the cisplatin-induced intrinsic AKI model, animals were given a single IP injection of cisplatin (5 mg/kg). All of the animals were euthanized 72 h after the first IP injection. Serum and urinary levels of Klotho, S100A8/A9, and NGAL were measured using an enzyme-linked immunosorbent assay. We also performed a proof-of-concept cross-sectional study to measure serum and urinary biomarkers in 61 hospitalized patients with established AKI. Compared to the intrinsic AKI group, the pre-renal AKI group showed a marked depression in urinary Klotho levels (13.21 ± 17.32 vs. 72.97 ± 17.96 pg/mL; P = 0.002). In addition, the intrinsic AKI group showed marked elevation of S100A8/A9 levels compared to the pre-renal AKI group (2629.97 ± 598.05 ng/mL vs. 685.09 ± 111.65 ng/mL; P = 0.002 in serum; 3361.11 ± 250.86 ng/mL vs. 741.72 ± 101.96 ng/mL; P = 0.003 in urine). There was no difference in serum and urinary NGAL levels between the pre-renal and intrinsic AKI groups. The proof-of-concept study with the hospitalized AKI patients also demonstrated decreased urinary Klotho in pre-renal AKI patients and increased urinary S100A8/A9 concentrations in intrinsic AKI patients. The attenuation of urinary Klotho and increase in urinary S100A8/A9 may allow differentiation between pre-renal and intrinsic AKI.

Soy isoflavone metabolism in cats compared with other species: urinary metabolite concentrations and glucuronidation by liver microsomes.

1. Soybean is a common source of protein in many pet foods. Slow glucuronidation of soy-derived isoflavones in cats has been hypothesized to result in accumulation with adverse health consequences. Here, we evaluated species' differences in soy isoflavone glucuronidation using urine samples from cats and dogs fed a soy-based diet and liver microsomes from cats compared with microsomes from 12 other species. 2. Significant concentrations of conjugated (but not unconjugated) genistein, daidzein and glycitein, and the gut microbiome metabolites, dihydrogenistein and dihydrodaidzein, were found in cat and dog urine samples. Substantial amounts of conjugated equol were also found in cat urine but not in dog urine. 3. ß-Glucuronidase treatment showed that all these compounds were significantly glucuronidated in dog urine while only daidzein (11%) and glycitein (37%) showed any glucuronidation in cat urine suggesting that alternate metabolic pathways including sulfation predominate in cats. 4. Glucuronidation rates of genistein, daidzein and equol by cat livers were consistently ranked within the lowest 3 out of 13 species' livers evaluated. Ferret and mongoose livers were also ranked in the lowest four species. 5. Our results demonstrate that glucuronidation is a minor pathway for soy isoflavone metabolism in cats compared with most other species.

BML-111 Attenuates Renal Ischemia/Reperfusion Injury Via Peroxisome Proliferator-Activated Receptor-α-Regulated Heme Oxygenase-1.

We examine whether BML-111, a lipoxin receptor agonist, inhibits renal ischemia/reperfusion (I/R) injury, and whether peroxisome proliferator-activated receptor-α (PPARα) or heme oxygenase-1 (HO-1) is involved in protective effects of BML-111 on kidney against I/R injury. Rats subjected to renal I/R injury were treated with or without BML-111. Renal histological and immunohistochemical studies were performed. Expressions of phosphorylated p38 mitogen-activated protein kinase (pp38 MAPK), phosphorylated PPARα (pPPARα), and HO-1 were assessed in NRK-52E cells exposed to BML-111. The binding activity of PPARα to peroxisome proliferator-responsive element (PPRE) on HO-1 promoter in the cells was determined. BML-111 treatment resulted in a marked reduction in the severity of histological features of renal I/R injury, and attenuated the rise in renal myeloperoxidase and malondialdehyde, blood urea nitrogen and creatinine, urinary N-acetyl-ß-glucosaminidase, and leucine aminopeptidase levels caused by I/R injury. BML-111 stimulated the renal expressions of pPPARα and HO-1, and cellular messenger RNA (mRNA) and protein expressions of pPPARα and HO-1 which were both blocked by GW6471, a selective PPARα antagonist, and ZnPP-IX, a specific inhibitor of HO-1 pretreatment. The pp38 MAPK inhibitor SB203580 blocked the BML-111-induced expressions of pp38 MAPK, pPPARα, and HO-1 in NRK-52E cells. The binding activity of PPARα to PPRE in nuclear extracts of NRK-52E cells was enhanced by treatment of the cells with BML-111, and was suppressed by GW6471 and SB203580. BML-111 protects the kidney against I/R injury via activation of p38 MAPK/PPARα/HO-1 pathway.

Urinary Klotho measured by ELISA as an early biomarker of acute kidney injury in patients after cardiac surgery or coronary angiography.

BACKGROUND: Acute kidney injury (AKI) is a common complication after cardiac surgery and percutaneous coronary interventions which markedly worsens prognosis. In recent years, new early biomarkers of AKI have been identified, but many important aspects still remain to be solved. Klotho is a pleiotropic protein that acts as a paracrine and endocrine factor in multiple organs. Reduced renal Klotho levels have been show in several animal models of AKI. No study has been published in which Klotho was tested in humans as an early marker of AKI. The aim of this work is to assess the usefulness of measuring urinary Klotho for the early diagnosis of AKI in patients with acute coronary syndrome or heart failure undergoing cardiac surgery or coronary angiography. METHODS: Urinary Klotho was measured 12 hours after intervention in 60 patients admitted to the Intensive Care Unit with acute coronary syndrome or heart failure secondary to coronary or valvular conditions, who underwent coronary angiography (30 patients) or cardiac bypass surgery or heart valve replacement (30 patients). The primary endpoint used was the onset of AKI according to the RIFLE classification system. Human Klotho levels were measured using an ELISA assay. RESULTS: We found no differences in urinary Klotho levels between AKI patients and those who did not develop AKI. Moreover, there was not significant correlation between urinary Klotho levels and the presence of AKI. CONCLUSION: Urinary Klotho measured by ELISA does not seem to be a good candidate to be used as an early biomarker of AKI.

Impact of Renal Transplantation and Nephrectomy on Urinary Soluble Klotho Protein.

BACKGROUND: Klotho is a single-pass transmembrane protein predominantly expressed in the kidneys. The soluble form of klotho has been shown to participate in various pathophysiological activities. However, information regarding the kinetics of soluble klotho remains limited. We herein assessed serial changes in the amounts of 24-hour urinary excreted soluble klotho among renal transplant recipients and concomitant living donors before and after transplantation. METHODS: A total of 15 recipients and donors were included in the current study, and the amounts of urinary soluble klotho were quantified using a sandwich enzyme-linked immunosorbent assay. RESULTS: Urine samples were available in 6 of the 15 recipients prior to the procedure. The amounts of urinary klotho in these 6 recipients and overall living donors at the baseline were 58.6 ng/day (IR: 29.3-142) and 698.8 ng/day (IR: 62.3-1619.5), respectively. Those in the recipients on postoperative day 2 (median 522.3 ng/day; IR 337.1-1168.5, P < .05) and day 5 (median 723.2 ng/day; IR 254.7-1238.6, P < .05) were significantly higher than the baseline values. Among the living donors, only a transient increase was observed in the amounts of urinary klotho on postoperative day 2. CONCLUSION: The current data regarding the urinary soluble klotho in recipients support the hypothesis that the kidney is a major source of urinary soluble klotho among the numerous components of the urinary tract. In living donors, the complex nature of events associated with acute reductions in the renal mass may modulate the release of soluble klotho from the kidneys into the urine.

Assessment of tubular reabsorption of phosphate as a surrogate marker for phosphate regulation in chronic kidney disease.

BACKGROUND/AIMS: Fibroblast growth factor 23 (FGF23) and soluble α-Klotho are emerging potential biomarkers of phosphorus and vitamin D metabolism which change in concentration in early chronic kidney disease (CKD) in order to maintain normal phosphorus levels. Tubular reabsorption of phosphate (TRP) has been commonly used to assess renal tubular phosphate transport. The aim of this study was to evaluate the usefulness of TRP as a surrogate marker of parameters of CKD-mineral bone disease (CKD-MBD) in CKD. METHODS: A cross-sectional study was performed in 93 stable patients with predialysis CKD stage 1-5. In all patients, TRP, estimated glomerular filtration rate (eGFR), calcium, phosphate, intact parathyroid hormone (iPTH), 25-hydroxyvitamin D, serum FGF23 and urine soluble α-Klotho levels were measured. RESULTS: As renal function declined, TRP significantly decreased (P < 0.001; r = 0.763) and both iPTH and serum FGF23 increased (P < 0.001; r = -0.598, P < 0.001; r = -0.453, respectively). The prevalence of hyperphosphatemia, secondary hyperparathyroidism, FGF23 excess and abnormal TRP increased progressively with declining eGFR. Although TRP level changed later than FGF23, abnormal levels of both TRP and FGF23 were observed earlier than changes in iPTH and serum phosphate. Decreased TRP was found to be independently associated with decreased eGFR and increased iPTH but was not associated with urine soluble α-Klotho or serum FGF23 level in multiple linear regression analysis. CONCLUSION: TRP is a simple, useful and cost-saving surrogate marker of the assessment of altered mineral metabolism in CKD patients and can be used as an alternative to serum FGF23, especially for mild to moderate renal insufficiency.

A rapid quantitative method for the analysis of synthetic cannabinoids by liquid chromatography-tandem mass spectrometry.

Synthetic cannabinoids represent an emerging drug problem in the USA, as these compounds are constantly being modified and rapidly sold as soon as they become available. Laboratories around the world are constantly improving the analytical methods to detect and identify these newly available designer drugs. This study used a simple approach to detect and quantify a variety of synthetic cannabinoids (14 parent compounds and 15 metabolites including series XLR, AM, JWH, UR, RCS, PB, HU and AB-FUBINACA) using LC-MS-MS. Drug-free urine samples spiked with various synthetic cannabinoids and their metabolites were separated on a C18-Hypersil Gold column using an Agilent 1290 ultra-high performance liquid chromatography and detected by an AB Sciex API 4000 tandem mass spectrometer. Studies were carried out to determine limit of detection, limit of quantitation, upper limit of linearity, ion suppression, interference, precision and accuracy to validate the method. Urine samples from patients and known users were hydrolyzed with ß-glucuronidase prior to the analysis by LC-MS-MS, and the data are presented. The method described here is rapid, highly sensitive and specific for the identification of a variety of synthetic cannabinoids.